WO2005106454A1 - Method and apparatus for detection of live bacterium within test subject antigen through specifically labeling thereof - Google Patents

Method and apparatus for detection of live bacterium within test subject antigen through specifically labeling thereof Download PDF

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Publication number
WO2005106454A1
WO2005106454A1 PCT/JP2005/003584 JP2005003584W WO2005106454A1 WO 2005106454 A1 WO2005106454 A1 WO 2005106454A1 JP 2005003584 W JP2005003584 W JP 2005003584W WO 2005106454 A1 WO2005106454 A1 WO 2005106454A1
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WIPO (PCT)
Prior art keywords
antigen
labeled
test
stationary phase
detection
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PCT/JP2005/003584
Other languages
French (fr)
Japanese (ja)
Inventor
Tohru Mikoshiba
Tetsuro Sasaki
Takaharu Enjoji
Original Assignee
Tohru Mikoshiba
Tetsuro Sasaki
Takaharu Enjoji
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Publication date
Application filed by Tohru Mikoshiba, Tetsuro Sasaki, Takaharu Enjoji filed Critical Tohru Mikoshiba
Priority to US11/587,846 priority Critical patent/US20070218500A1/en
Priority to JP2006512729A priority patent/JPWO2005106454A1/en
Publication of WO2005106454A1 publication Critical patent/WO2005106454A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/52—Sorbents specially adapted for preparative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/58—Use in a single column

Definitions

  • the present invention relates to a detection method and a detection device for detecting the concentration or number of an antigen in a solution, and in particular, to speed up a test by specifically labeling live bacteria in an antigen.
  • TECHNICAL FIELD The present invention relates to a detection method and a detection device capable of speeding up.
  • the amount of an antigen is measured by using a change in a physical quantity or the like due to an antigen-antibody reaction without binding a labeling substance such as an enzyme to the antibody, such as a surface plasmon resonance method.
  • Antibodies such as unlabeled immunochromatography and radioimmunoassay
  • the amount of antigen is measured by measuring the amount of the labeling substance using a substance bound with an enzyme or other labeling substance.
  • the "sandwich method (sandwich ELISA method)" which is currently the mainstream because the measurement operation is relatively easy, will be described in detail.
  • FIG. 12 is a schematic view showing main steps of a conventional sandwich method.
  • A is a step of immobilizing an antibody (primary antibody)
  • (b) is a step of capturing a target bacterium (antigen)
  • (c) is a step of staining with an enzyme-labeled antibody
  • (d) is a step of staining with an enzyme-labeled antibody.
  • This is the step of immobilizing the antibody (secondary antibody)
  • (e) is the step of eluting the labeled iridani cells
  • (f) is the step of detecting the eluted labeled iridani cells.
  • FIGS. 12A to 12F show the surface 100 of the immobilization layer, the primary antibody 101, the target bacterium 102, the secondary antibody 103, the labeling substance 104, the light source 105, and the detector 106. .
  • a solution containing a primary antibody 101 that specifically binds to pathogenic Escherichia coli O—157 is poured into a reaction vessel in which non-specific adsorption is likely to occur.
  • the primary antibody 101 is non-specifically adsorbed on the surface 100 of the immobilization layer to immobilize it (FIG. 12 (a)).
  • a sample solution containing the target bacterium 102 is poured into the reaction vessel, and the target bacterium 102 is specifically bound to the immobilized primary antibody 101 in the reaction vessel by an antigen-antibody reaction (FIG. 12 (b)).
  • a solution containing the secondary antibody 103 labeled with the labeling substance 104 is poured into a reaction vessel, and the enzyme-labeled antibody comprising the secondary antibody 103 and the labeling substance 104 is reacted. Specific binding is achieved by an antigen-antibody reaction via the target bacterium 102 in the container (FIG. 12 (c)). As a result, an enzyme-labeled antibody in an amount proportional to the target bacterium 102 can be immobilized on the reaction vessel (FIG. 12 (d)).
  • a substrate solution containing a coloring substrate is added to the reaction vessel, and the enzyme-labeled antibody is colored by an enzymatic reaction.
  • the target bacterium 102 bound with the enzyme-labeled antibody is eluted with a lysis extract such as an aqueous sodium hydroxide solution (FIG. 12 (e)), and then placed opposite the light source 105.
  • the detected detector 106 detects light having a wavelength that the dye specifically absorbs, and measures the antigen concentration (FIG. 12 (f)).
  • Patent Document 1 discloses a method in which a stationary phase in which a specific binding component capable of specifically binding to Escherichia coli is immobilized is applied to an arbitrary region on the surface of the water-absorbing substrate.
  • a detection kit comprising at least a step of developing a substrate solution.
  • the detection method and the detection kit by appropriately adjusting the water absorption of the water-absorbent substrate, the speed at which the test liquid and the substrate liquid are developed can be optimized, and thus, the inspection speed can be improved. It is possible to make a quick dagger.
  • the chromogenic substrate component specifically binds to the alkaline phosphatase of the live bacteria captured on the stationary phase by the specific binding component to form a color. Therefore, there is an advantage that it is possible to detect even the presence or absence of viable E. coli in the test solution.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2002-165599 (paragraph number [0033] [0037])
  • Patent Document 1 the detection method described in Patent Document 1 can speed up the test and detect live bacteria of Escherichia coli, but can handle only a very small amount of sample of 0.2 Olml to 0.2 ml. Therefore, there is a problem that the reliability of the inspection cannot be ensured (see Paragraph No. [0034] of Patent Document 1). In other words, when the amount of the sample to be tested is small, the probability of containing E. coli and the probability of containing viable bacteria in E. coli also decrease. It will be low.
  • the present invention has been made in view of the above points, and an object of the present invention is to enable rapid detection of viable bacteria among microorganisms serving as antigens in a short time, and to increase the reliability of the test. Another object of the present invention is to provide a detection method and a detection device that can secure the same.
  • the present invention provides a test antigen such as Escherichia coli with a labeled antigen obtained by allowing a labeling substance which is enzymatically decomposed by viable bacteria in the test antigen to act on the antigen. After generation, the labeled antigen is captured in a stationary phase in which a specific binding antibody capable of specifically binding to the test antigen is immobilized.
  • the present invention provides the following.
  • test antigen and a labeling substance which is enzymatically decomposed by the live bacteria in the test antigen specifically labels and detects the live bacteria in the test antigen.
  • a detection method wherein the labeled substance is allowed to act on the test antigen to generate an optically detectable labeled antigen, and a specific binding antibody capable of specifically binding to the test antigen is immobilized.
  • a detection method comprising capturing the labeled antigen in a stationary phase.
  • the activity of a test antigen such as Escherichia coli and a labeled substance which is enzymatically degraded by the live bacteria among the test antigens is evaluated.
  • This is a detection method for detecting labeled antigens, in which a labeled antigen is allowed to act on a test antigen to generate a labeled antigen that can be optically detected by a fluorescence reaction or the like, and the antigen-antibody reaction specifically reacts with the test antigen.
  • the capture targets are an optically detectable labeled antigen (live bacteria) and a dead bacterium (fungal fragment) that is not labeled and cannot be detected optically.
  • the conventional sandwich method does not require an essential secondary antibody, and as a result, the antigen-antibody reaction, which was required twice in the past, can be performed only once. Can be.
  • the only optically detectable antigen is a live bacterium serving as the labeling antigen, so that it is possible to detect live bacteria Z and dead bacteria separately. Viable bacteria causing food poisoning damage can be accurately detected.
  • the amount of sample handled by the detection method according to the present invention is several tens ml- Since the sample volume is several hundred ml, it is possible to prevent a decrease in the probability of containing the test antigen due to sample extraction, thereby preventing a decrease in detection accuracy and detection sensitivity and ensuring test reliability. it can.
  • a detection method comprising capturing the labeled antigen grown by a growth medium in the stationary phase.
  • the labeled antigen grown in the growth medium is captured, so that the concentration of the labeled antigen can be increased as compared to before the addition of the growth medium.
  • the probability of capturing the labeled antigen can be improved, and the detection accuracy and the detection sensitivity can be increased.
  • a detection method comprising capturing the circulated labeled antigen in the stationary phase while circulating the sample solution containing the labeled antigen a plurality of times.
  • the circulating labeled antigen is captured in the stationary phase while the sample solution containing the labeled antigen is circulated a plurality of times.
  • the sample solution can be brought into contact with the immobilized stationary phase a plurality of times, so that the probability of capturing the labeled antigen can be improved, and the detection accuracy and the detection sensitivity can be improved.
  • test antigen is captured in a plurality of stationary phases formed by immobilizing a specific binding antibody capable of specifically binding to each of the plurality of test antigens.
  • a detection method characterized in that:
  • the test antigens are collectively captured at one time in a series of test flows, the test can be speeded up and efficiency can be improved.
  • a detection device having a column capable of installing a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen, wherein the column is provided with the stationary phase. 5. A detection device, wherein the test antigen captures a labeled antigen labeled.
  • a column in which a stationary phase formed by immobilizing a specific conjugate capable of specifically binding to a test antigen (including live bacteria and dead bacteria) such as Escherichia coli can be installed.
  • a column on which the stationary phase described above is installed captures labeled antigens in which the test antigen is labeled by the labeled antigens acting only on viable bacteria.
  • a single antigen-antibody reaction is sufficient before capturing the targeted antigen, which enables rapid testing and enables accurate detection of viable bacteria causing food poisoning damage.
  • the detection device further comprising a stirring device for stirring the liquid, wherein the labeled antigen is labeled by the stirring device.
  • the above-described detection device further includes a stirring device for mechanically stirring the liquid (sample solution), and the above-described labeled antigen is added to the labeling antigen in the stirring device. Therefore, the labeling of the test antigen can be promoted, and the labeled antigen can be efficiently produced.
  • the above-mentioned column can be used a plurality of times, a plurality of microbiological tests can be continuously and efficiently performed.
  • a detection device having a plurality of columns capable of installing a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen, wherein the stationary phase is installed.
  • a detection device having a plurality (a plurality of types) of columns on which a stationary phase formed by solidifying a specific binding antibody capable of binding specifically to a test antigen can be provided. Since the labeled antigen in which the test antigen is labeled is captured by using a plurality of columns provided with a stationary phase, the test can be performed quickly and efficiently.
  • a biocolumn provided with a stationary phase in which a specific binding antibody capable of specifically binding to a test antigen is immobilized.
  • the test can be further speeded up. 'I can make sure.
  • a storage means such as a freeze-drying method, the biocolumn can be stored in a stable state for a long time.
  • the antigen-antibody reaction which was conventionally required twice, can be performed only once, so that the test can be performed quickly. Since the antigens to be detected are labeled antigens (live bacteria), it is possible to distinguish between live bacteria and dead bacteria. Furthermore, since a large number of samples can be handled, the reliability of the test can be ensured. You can do it.
  • FIG. 1 is an external view of a detection device 1 according to an embodiment of the present invention.
  • a detection device 1 has a pump and a valve inside.
  • a biocolumn 2 provided with a stationary phase for capturing a target bacterium is provided on a side surface of a box-shaped control box having the following.
  • An M-Cell 3 containing a lysate that elutes the captured target bacterium is installed next to the No. 2 column.
  • FIG. 2 is an enlarged view of the biocolumn 2 installed in the detection 1 according to the embodiment of the present invention.
  • the biocolumn 2 is prepared by filling glass beads for a biocolumn into glass beads capable of capturing an antigen by an antigen-antibody reaction.
  • the glass beads are pre-treated with an aqueous sodium hydroxide solution or hydrochloric acid, and then dried overnight. Then, the glass beads are subjected to a sintering treatment and a silyllizing treatment with a silylating agent, rinsed, and dried at room temperature to produce silylated glass beads.
  • silylated glass beads is filled in a glass tube for a biocolumn. Then, it is immersed for several minutes in a dartartaldehyde solution containing dartartaldehyde as a coupling agent, washed with a phosphate buffer solution, and then subjected to a primary antibody immobilization treatment by non-specific adsorption. In the primary antibody immobilization treatment, the unreacted primary antibody is removed by appropriately washing the glass tube for the biocolumn.
  • a blocking solution containing sialic albumin serum as a blocking agent is injected, and the blocking agent is non-specifically applied to the non-specific adsorption surface remaining on the surface of the glass beads. Adsorb and prevent subsequent non-specific adsorption of other organic substances.
  • the inside of the glass tube for the biocolumn is washed a plurality of times with a phosphate buffer or the like to remove the unreacted blocking agent, whereby the Noocolumn 2 is produced.
  • a storage means such as a freeze-drying method, for example, the biocolumn 2 can be stored in a stable state for a long time.
  • the present invention is not limited to this.
  • a silylation treatment, a coupling treatment, etc. Uses relatively easy-to-handle flat glass to establish various conditions It may be good.
  • spherical glass beads are used.
  • glass beads are one of carriers for immobilizing antibodies (a matrix for immobilizing antibodies), and are used for immobilizing antibodies.
  • the carrier may have any shape as long as the carrier has a surface area as much as possible and the antibody and the sample can be sufficiently brought into contact with the sample in a state where the column is packed.
  • the production process is almost the same as the production process of the Noo column 2 using the glass beads described above, and thus the description thereof is omitted.
  • the present invention is applicable irrespective of the material of the beads, the silylation treatment conditions, and the method of immobilizing the primary antibody, as long as the primary antibody can be immobilized on the surface of the immobilization layer.
  • FIG. 3 is a flow path system diagram when performing a microorganism test using the detection device 1 shown in FIG.
  • FIG. 4 is a flowchart schematically showing an inspection process in the flow path diagram shown in FIG.
  • 6-carboxylfluorescein diacetate, a dilution (CFDA dilution) solution solution containing a labeling substance
  • CFDA dilution a dilution solution
  • a test sample for example, 100 ml
  • a test antigen containing a mixture of live bacteria (labeled antigen) and dead dead bacteria that have not been hydrolyzed is injected into bottles B5 and B6.
  • a phosphate buffer solution as a column washing solution is injected, and an alkaline aqueous solution that elutes target bacteria captured in the biocolumn 2 is injected into M-Cell3.
  • CFDA used in the present embodiment can be used as an agent capable of staining viable bacteria and detecting them by fluorescence or the like.
  • Step Sl the fixing step is performed (Step Sl). More specifically, in Fig. 3, by operating the pump P1, the addition test of the CFDA diluent injected into the bottle B1 changes the bottle Bl ⁇ valve Vl ⁇ valve V2 ⁇ pump Pl ⁇ valve 3 ⁇ biocolumn. Flows in the order of 2 ⁇ valve 4 ⁇ valve 5 ⁇ valve 6 ⁇ bottle B2. The required time is about 15 minutes.
  • the sampled water stored in bottle B2 and flowing into the bottle B2 is detected as bottle B2 ⁇ valve Vl ⁇ valve V2 ⁇ pump Pl ⁇ valve V3 ⁇ biopower ram 2 ⁇ valve 4 ⁇ valve 5 ⁇ valve 6 ⁇ Flow in the order of bottle B3. This takes about 15 minutes.
  • the biocolumn 2 glass
  • the test antigen in the sample is specifically bound to the primary antibody immobilized on the beads by an antigen-antibody reaction.
  • a growth culture solution may be added to the bottle B1, and the labeled antigen grown by the growth culture solution may be captured. Thereby, the concentration of the labeled antigen can be increased, and the probability of capturing the labeled antigen can be improved.
  • the stationary phase (glass beads) in which the specific binding antibody is immobilized and the circulating sample solution (sample) are sufficiently used. They must be in contact. Therefore, in this embodiment, the glass beads (stationary phase) in the Noo column 2 are effectively agitated using an electromagnetic pinch valve PV (see FIG. 3)! More specifically, this will be described with reference to FIG. FIG. 5 is an explanatory diagram showing how the stationary phase is effectively stirred.
  • the pinch valve PV is opened and closed at a predetermined timing, so that the stationary phase (glass beads) is periodically and effectively. It is configured to be stirred. This makes it possible to bring the stationary phase (glass beads) into contact with the sample sufficiently.
  • the electromagnetic pinch valve PV is used.
  • the present invention is not limited to this.
  • a manual or electric pinch valve may be used.
  • any means may be used as long as the stationary phase in the biocolumn 2 has an effect of being appropriately stirred, and is not particularly limited to a pinch valve.
  • a cleaning step is performed (Step S2). More specifically, in Figure 3, by switching the valve V2, the phosphate buffer power stored in bottle B5 bottle 5 ⁇ valve 2 ⁇ pump Pl ⁇ valve V3 ⁇ biocolumn 2 ⁇ valve V4 ⁇ Flow in the order of valve V5 ⁇ bottle 4. Then, switch off the pump P1. It takes about 15 minutes.
  • the washing step in step S2 the phosphate buffer is passed through the biocolumn 2 to wash unreacted primary antibodies and the like, and condensate the test antigen as a result.
  • Step S3 an elution step is performed (Step S3). More specifically, in FIG. 3, by switching valve V3 and valve V4 and activating M-Cell3 containing the lysate for eluting the antigen to be tested and pump P2, the sample captured in biocolumn 2 was changed. Elute the test antigen. Then, a fluorescent spectrophotometer equipped with a flow cell (lower right in FIG. 3) optically detects (spectrum measurement) labeled live bacteria (labeled antigen) among the test antigens. Through the elution step of step S3, only viable bacteria causing food poisoning damage are detected. Then, the microbial examination is temporarily terminated by a series of steps from step S1 to step S3.
  • step S4 When a microbe test is continuously performed by injecting a chemical solution for several experiments into bottle B4 or bottle B6, a washing step is additionally performed as shown in Fig. 4 (step S4). More specifically, in FIG. 3, the pump P2 is operated, the valve V4 and the valve V7 are switched, and the biocolumn 2 is washed with the phosphate buffer stored in the bottle B6.
  • Step S4 As described above, according to the series of detection steps from Step S1 to Step S3 (Step S4) shown in FIG. 4, it can be seen that only viable bacteria can be detected from microorganisms as antigens.
  • the amount of water that can be tested by the detection device 1 according to the embodiment of the present invention is different from the amount of water to be used in a test kit or the like (about 0.2 ml—0.2 ml), and several tens ml— Since it is several hundred ml, it is possible to prevent a decrease in the probability of containing Escherichia coli caused by sample siding of the sample, and to improve detection accuracy and detection sensitivity.
  • the chemicals used in each of the steps of washing, elution, and washing and the method thereof can be changed without departing from the spirit of the present invention.
  • step S4 the microorganism test can be performed in a shorter time than the conventional sandwich method. Detector
  • a quick test using the method 1 with reference to the schematic diagram of FIG.
  • FIG. 6 is a schematic diagram showing main steps of the detection method according to the embodiment of the present invention.
  • (a) is the step of immobilizing the antibody (primary antibody)
  • (b) is the step of stirring the sample solution containing the test antigen and the fluorescent reagent
  • (c) is the test antigen containing the labeled antibody.
  • (D) is the step of immobilizing the test antigen
  • (e) is the step of eluting the test antigen
  • (f) is the step of immobilizing the labeled antigen among the eluted test antigens. This is a detection step for detecting only the antigen.
  • 6 (a)-(f) show the surface of the immobilization layer 10, the primary antibody 11, the target bacteria (live bacteria) 12, the labeled substance 13, the labeled antigen 14, the light source 15, and the detector 16. Has been described.
  • the primary antibody 11 is non-specifically adsorbed on the surface 10 of the immobilization layer of the glass beads filled in the glass column for a Noyo column, and immobilization is performed (FIG. 6 (a)).
  • the details of this step are as described in the preparation step of the biocolumn 2.
  • the bacterium 12 as a target bacterium is caused to emit light (FIG. 6 (b)). More specifically, when the diluted CFDA solution is added to the sample solution, live bacteria among the test antigens absorb CFDA (labeling substance 13), which is an intracellular pH indicator, and hydrolyze. It becomes fluorescent by decomposition. That is, CFDA has a function as a viable stain. After the CFDA diluent is added to the sample solution, the hydrolysis by viable bacteria may be promoted by stirring with a stirrer. As a result, CFDA can be absorbed into living bacteria in a shorter time and more reliably, thereby contributing to quick inspection.
  • CFDA labeling substance 13
  • a sample solution containing the test antigen (including the labeled antigen 14) is brought into contact with the surface 10 of the immobilized layer of the biocolumn 2, and the test solution is subjected to an antigen-antibody reaction with the primary antibody 11. Capture the antigen (Fig. 6 (c)). After capturing the test antigen, a washing solution such as a phosphate buffer is poured into the biocolumn 2 to remove impurities, unreacted primary antibodies, etc., thereby condensing (concentrating) the test antigen and The test antigen is fixed (Fig. 6 (d)).
  • test antigen immobilized by the primary antibody 11 and containing the labeled antigen 14 is lysed and extracted with an aqueous solution of Arikari (FIG. 6 (e)).
  • concentration in the lysate extract can be increased by reducing the volume in the circulation path and the flow cell, and by reducing the amount of alkaline aqueous solution required for lysis * extraction, thereby improving detection sensitivity. be able to.
  • a test antigen can be lysed and extracted more quickly and reliably by using a force using a high-concentration alkaline aqueous solution, for example, an acidic buffer solution / surfactant in combination. .
  • the labeled antigen 14 is optically detected by the detector 16 arranged opposite to the light source 15 (FIG. 6 (f)). More specifically, the labeled antigen 14 containing the labeling substance 13 emits fluorescent light by ultraviolet excitation light emitted from the light source 15 and is received by the detector 16 having a condenser lens to generate an electric signal. (Chromatographic signal). By measuring and analyzing the electric signal, the labeled antigen 14 (target bacterium 12) can be optically detected.
  • the detection mode does not matter, for example, a force using a fluorescence spectrophotometer.
  • a detector such as a particle counter is used.
  • a microorganism test can be performed in a shorter time than in the conventional sandwich method. That is, in the conventional San Deutsch method, two antigen-antibody reactions were required before detection of the test antigen (see FIGS. 12 (b) and 12 (c)). Since only one antigen-antibody reaction between the antigen 14 and the primary antibody 11 is required (see FIG. 6 (c)), the test time can be reduced by that much, and the test can be performed quickly. Can be planned.
  • FIG. 7 is an external view of a detection device according to another embodiment of the present invention.
  • the main feature is that two Noo columns 65 and 66 that can capture specific target bacteria are provided.
  • the force of providing two biocolumns 65 and 66 is not limited to this.
  • three or more biocolumns may be provided.
  • Providing multiple biocolumns enables simultaneous detection of multiple types of target bacteria
  • each device such as a 'pump' bottle is installed inside a constant temperature bath (square frame in the drawing) at 35 ⁇ 1 ° C.
  • the respective devices and pumps are optimally controlled by the flow path control sequencer 69.
  • a test sample supply tank containing the target bacteria (sample hopper) 61, a stirrer for stirring the sample (magnetic stirrer) 62, a filtration filter 63 for removing impurities, and a flow path switch for switching the flow paths appropriately
  • a valve 64, biocolumns 65 and 66 filled with fine particle glass with the target bacteria antibody fixed on the surface, a circulating pump 67 for flowing the sample, and a high-sensitivity fluorescence detector 68 for optically detecting the target bacteria are installed.
  • a bottle B11 containing a washing solution, a bottle B12 containing a fixative solution, and a bottle B13 containing a lysate of a captured stained bacterium are provided.
  • the inspection process using the detection device shown in FIG. 7 will be outlined.
  • a specified amount of a sample is striked by a standard method, and a test solution (50 to 100 ml) is put into a test sample supply tank 61. Then, while stirring with the stirrer 62, CFDA as a fluorescent staining reagent is added to stain live bacteria. After stirring for about 10 minutes, the impurities are removed through the filtration filter 63 and introduced into the sample channel (biocolumn 65). Switch the flow path switching valve 64 to the filter back washing system, and wash the filtration filter 63.
  • the test solution that has passed through the filtration unit passes through the biocolumns 65 and 66, and circulates through all the washing channels in the biocolumns 65 and 66. Pass through.
  • the stained bacterial cells (labeled antigen) captured by the immobilized antibody were re-used several times by recycling the lysed extract added in small amounts to the recycling channel in the biocolumns 65 and 66 using the bottle B13. Extracted to high concentration.
  • the test solution from which extraction has been completed is introduced into the high-sensitivity fluorescence detector 68 by the switching valve 64 below the biocolumns 65 and 66, and a chromatogram is drawn as an electric signal.
  • the test solution from which extraction has been completed is introduced into the flow cell of the high-sensitivity fluorescence detector 68, and the stained bacterial cells in the sample solution are fluoresced by ultraviolet excitation light emitted from the light source. Emits. Then, the fluorescent light is received by a condenser lens, and an optical signal is converted into an electric signal, whereby a chromatogram is drawn.
  • the detection method according to the embodiment of the present invention can sufficiently detect a target bacterium without a culturing step. However, more reliable test results can be obtained by culturing the target bacteria as necessary.
  • the culturing step is performed, for example, by providing a heater in the detection device and performing culturing before the fixing step, or by culturing the entire detection device in which the inspection process is incorporated at a constant temperature. can do.
  • FIG. 8 is a diagram showing the measurement results when a performance experiment of the biocolumn 2 was performed in the flow channel system shown in FIG. More specifically, it shows the CFDA fluorescence intensity with respect to the injection amount (CFUZlOOml) of E. coli.
  • CFUZlOOml injection amount
  • FIG. 8 shows the CFDA fluorescence intensity with respect to the injection amount (CFUZlOOml) of E. coli.
  • CFUZlOOml injection amount
  • FIG. 10 is a table showing measurement results when a performance experiment of the biocolumn 2 was performed in the flow channel system shown in FIG.
  • FIG. 10 (a) shows the CFDA fluorescence intensity for the dead bacteria subjected to the staining treatment.
  • the CFDA fluorescence intensity in the lysed extract was very weak. That is, even if dead bacteria that do not directly cause food poisoning are mixed in the actual test water, viable bacteria can be evaluated with high accuracy without any interference, and only dead bacteria can be detected. It can be seen that the problem of eliminating conforming products, including, for example, can be solved.
  • Fig. 10 (b) shows CFDA versus injection amount (CFUZlOOml) of multiple types of bacteria other than E. coli (E. coli: C. freundii, Enterobacteriaceae: S. marcescens). Shows the fluorescence intensity. According to the table shown in FIG. 10, it can be seen that the lysis extract has a low CFDA fluorescence intensity for bacteria other than E. coli. In other words, similarly to dead bacteria, even when bacteria other than the target bacteria coexist in the sample solution, the effect can be relatively low.
  • FIG. 11 is a table showing measurement results when performance tests of the biocolumn 2 by repeated use were performed in the flow channel system shown in FIG. More specifically, it shows the CFDA fluorescence intensity with respect to the cumulative number of uses (times) of the biocolumn 2.
  • FIG. 11 it can be seen that when the biocolumn 2 is used twice cumulatively, the CFDA fluorescence intensity in the lysed extract decreases by about 98% at the second use. This is because a high concentration of alkali having a lytic action is used for extraction of the target bacterium.
  • the antibody which is a protein, is damaged together with the bacterium. Therefore, for example, by using a lysate extract that causes less damage to the antibody, the biocolumn 2 can be used multiple times.
  • 100 ml of the bacterial solution whose number of bacteria is estimated by the MPN method or the like is transferred to a sample supply bottle of a test device, and 1 ml of a viable bacterial staining solution containing CFDA is added. After circulating twice through the biocolumn, drain. Then, after applying the entire amount of the sample solution to the biocolumn, an appropriate amount of the biocolumn washing solution is flowed, and after the inside of the biocolumn is washed, the flow path is switched and all the biocolumn washing solution remaining inside the biocolumn is removed.
  • the target bacteria stained with live bacteria in the above-described steps and trapped in the biocolumn were lysed and extracted using a lysis extract of a total amount of 1 Oml, and introduced into the flow cell of the fluorescence spectrophotometer. Measurement. Then, after the measurement is completed, all the channel systems are washed with sterile phosphate buffered diluted water V.
  • the detection method and the detection device according to the present invention detect a labeled antigen obtained by allowing a labeled substance that is enzymatically decomposed by viable bacteria in the test antigen to act on the test antigen, thereby detecting the sample.
  • the viable bacteria in the solution can be used as target bacteria, and are useful as those that can ensure quickness and certainty of the test.
  • FIG. 1 is an external view of a detection device according to an embodiment of the present invention.
  • FIG. 2 is an enlarged view of a biocolumn installed in the detection device according to the embodiment of the present invention.
  • FIG. 3 is a flow path diagram when performing a microorganism test using the detection device shown in FIG. 1.
  • FIG. 4 is a flowchart showing an outline of an inspection process in the flow path diagram shown in FIG. 3.
  • FIG. 5 is an explanatory view showing how a stationary phase is effectively stirred.
  • FIG. 6 is a schematic view showing main steps of a detection method according to an embodiment of the present invention.
  • FIG. 7 is an external view of a detection device according to another embodiment of the present invention.
  • FIG. 8 is a view showing measurement results when a performance experiment of a biocolumn was performed in the flow channel system shown in FIG. 3.
  • FIG. 9 is a diagram in which each data of the table shown in FIG. 8 is plotted in a two-dimensional field.
  • FIG. 10 is a table showing measurement results when performing a performance experiment on a biocolumn in the flow channel system shown in FIG. 3.
  • FIG. 11 is a table showing measurement results of performance tests of a biocolumn by repeated use in the flow channel system shown in FIG. 3.
  • FIG. 12 is a view showing main steps of a conventional sandwich method.

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Abstract

A method and apparatus for detection whereby live bacteria among microbes as an antigen can be detected rapidly in a short period of time through specifically labeling of live bacteria within a test subject antigen and whereby testing assurance can be ensured. The method and apparatus are characterized in that labeled antigen (14) is formed by action, on a test subject antigen such as Escherichia coli, of labeled substance (13) zymolyzable by live bacteria (target bacteria (12)) within the test subject antigen, and the resultant labeled antigen (14) is trapped on an immobilization phase having, immobilized thereon, a specific binding antibody capable of specifically binding to the test subject antigen.

Description

明 細 書  Specification
被検抗原内の生菌を特異的に標識化して検出する検出方法及び検出装 置  Detection method and detection device for specifically labeling and detecting live bacteria in test antigens
技術分野  Technical field
[0001] 本発明は、溶液中の抗原の濃度や数を検出する検出方法及び検出装置に関する ものであって、特に、被抗原内の生菌を特異的に標識ィ匕することによって検査の迅 速ィ匕を図ることが可能な検出方法及び検出装置に関するものである。  The present invention relates to a detection method and a detection device for detecting the concentration or number of an antigen in a solution, and in particular, to speed up a test by specifically labeling live bacteria in an antigen. TECHNICAL FIELD The present invention relates to a detection method and a detection device capable of speeding up.
背景技術  Background art
[0002] 近年、サルモネラ菌、ブドウ球菌、ボツリヌス菌、病原性大腸菌 O— 157といった微 生物に起因する食中毒の被害が問題になっており、関係企業では、これらの微生物 に対する予防 '衛生にかかわる講習会や啓蒙活動などを行う一方で、高額な設備投 資を通じて事故拡散を未然に防ごうとしている。  [0002] In recent years, food poisoning caused by microorganisms such as Salmonella, Staphylococcus, Clostridium botulinum, and pathogenic Escherichia coli O-157 has become a problem. While conducting educational and educational activities, it is trying to prevent the spread of accidents through expensive capital investment.
[0003] 微生物の検出は、培養した後に種類の同定や定量をすることが一般的である。す なわち、前培養→増菌培養→分離培養といった培養操作を伴うことから、その培養 操作に起因して検査結果が出るまで数日程度の期間を要し、かつ、専門の測定技術 者を必要とする。この長期間の測定は、迅速性が要求される生鮮食品など、食料品 への微生物検査の必要性が生じた場合に非常に問題となる。  [0003] In the detection of microorganisms, it is common to identify or quantify the type after culturing. In other words, since a culture operation such as pre-culture → enrichment culture → separation culture is involved, it takes a few days for the test results to be obtained due to the culture operation, and a specialized measurement engineer is required. I need. This long-term measurement can be very problematic when a need arises for microbiological testing of foodstuffs, such as fresh foods, which require speed.
[0004] このようなことから、食中毒病原菌を簡易かつ迅速に検出する様々な試薬や装置が 提案されている。例えば、免疫化学反応を応用し、特定の菌 (抗原)と特異的に結合 する抗体を用いてその特定の菌を凝集させ、抗原濃度を測定'分析する免疫クロマト グラフ法が広く知られている。以下、この免疫クロマトグラフ法について、体表面に固 有の抗原決定基を有して 、る病原性大腸菌 O - 157 (O - 157と 、う抗体が特異的に 結合する抗原決定基を菌体表面に発現した病原性を有する大腸菌)を抗原の一例 に採り、詳説する。  [0004] Under such circumstances, various reagents and devices for simply and quickly detecting food poisoning pathogens have been proposed. For example, an immunochromatography method that applies an immunochemical reaction to agglutinate a specific bacterium using an antibody that specifically binds to the specific bacterium (antigen) and measures the antigen concentration 'analysis is widely known. . Hereinafter, in this immunochromatography, the pathogenic Escherichia coli O-157 (O-157, which has a specific antigenic determinant on the body surface) Escherichia coli (pathogenic Escherichia coli) expressed on the surface is taken as an example of the antigen and will be described in detail.
[0005] なお、免疫クロマトグラフ法には、例えば表面プラズモン共鳴法など、抗体に酵素な どの標識物質を結合せずに、抗原抗体反応による物理量などの変化を利用して抗 原の量を測定する非標識免疫クロマトグラフ法と、ラジオィムノアッセィ法など、抗体 に酵素などの標識物質を結合させたものを使用し、標識物質の量を測定することで 抗原の量を測定する標識免疫クロマトグラフ法とがあるが、ここでは後者の標識免疫 クロマトグラフ法について、特に、測定操作が比較的容易であることから現在主流とな つて 、る「サンドイッチ法 (サンドイッチ ELISA法）」につ 、て詳説する。 [0005] In immunochromatography, for example, the amount of an antigen is measured by using a change in a physical quantity or the like due to an antigen-antibody reaction without binding a labeling substance such as an enzyme to the antibody, such as a surface plasmon resonance method. Antibodies, such as unlabeled immunochromatography and radioimmunoassay There is a labeled immunochromatography method in which the amount of antigen is measured by measuring the amount of the labeling substance using a substance bound with an enzyme or other labeling substance. In particular, the "sandwich method (sandwich ELISA method)", which is currently the mainstream because the measurement operation is relatively easy, will be described in detail.
[0006] 図 12は、従来のサンドイッチ法の主要工程を示す模式図である。（a)は抗体 (一次 抗体)の固定ィ匕工程であり、（b)は標的菌 (抗原)の捕捉工程であり、（c)は酵素標識 化抗体による染色工程であり、（d)は抗体 (二次抗体)の固定ィ匕工程であり、（e)は標 識ィ匕菌体の溶出工程であり、（f)は溶出させた標識ィ匕菌体の検出工程である。なお、 図 12 (a)—（f)には、固定化層表面 100、一次抗体 101、標的菌 102、二次抗体 10 3、標識化物質 104、光源 105、検出器 106が記載されている。  [0006] Fig. 12 is a schematic view showing main steps of a conventional sandwich method. (A) is a step of immobilizing an antibody (primary antibody), (b) is a step of capturing a target bacterium (antigen), (c) is a step of staining with an enzyme-labeled antibody, and (d) is a step of staining with an enzyme-labeled antibody. This is the step of immobilizing the antibody (secondary antibody), (e) is the step of eluting the labeled iridani cells, and (f) is the step of detecting the eluted labeled iridani cells. FIGS. 12A to 12F show the surface 100 of the immobilization layer, the primary antibody 101, the target bacterium 102, the secondary antibody 103, the labeling substance 104, the light source 105, and the detector 106. .
[0007] 図 12において、まず、非特異的吸着が起こりやすい反応容器に、病原性大腸菌 O — 157 (標的菌 102)に特異的に結合する一次抗体 101を含む溶液を注ぎ、反応容 器の固定ィ匕層表面 100に一次抗体 101を非特異吸着させて固定ィ匕する（図 12 (a) ) 。そして、反応容器に標的菌 102を含む試料溶液を注ぎ、標的菌 102を反応容器内 の固定ィ匕された一次抗体 101に抗原抗体反応で特異的に結合させる（図 12 (b) )。  [0007] In FIG. 12, first, a solution containing a primary antibody 101 that specifically binds to pathogenic Escherichia coli O—157 (target bacterium 102) is poured into a reaction vessel in which non-specific adsorption is likely to occur. The primary antibody 101 is non-specifically adsorbed on the surface 100 of the immobilization layer to immobilize it (FIG. 12 (a)). Then, a sample solution containing the target bacterium 102 is poured into the reaction vessel, and the target bacterium 102 is specifically bound to the immobilized primary antibody 101 in the reaction vessel by an antigen-antibody reaction (FIG. 12 (b)).
[0008] 次に、反応容器に、標識ィ匕物質 104によって標識ィ匕された二次抗体 103を含む溶 液を注ぎ、二次抗体 103と標識化物質 104とからなる酵素標識化抗体を反応容器内 の標的菌 102を介して抗原抗体反応で特異的に結合させる（図 12 (c) )。これにより 、標的菌 102に比例した量の酵素標識ィ匕抗体を反応容器に固定ィ匕することができる (図 12 (d) )。また、発色基質を含む基質溶液を反応容器に添加し、酵素反応によつ て酵素標識化抗体を発色させておく。  Next, a solution containing the secondary antibody 103 labeled with the labeling substance 104 is poured into a reaction vessel, and the enzyme-labeled antibody comprising the secondary antibody 103 and the labeling substance 104 is reacted. Specific binding is achieved by an antigen-antibody reaction via the target bacterium 102 in the container (FIG. 12 (c)). As a result, an enzyme-labeled antibody in an amount proportional to the target bacterium 102 can be immobilized on the reaction vessel (FIG. 12 (d)). In addition, a substrate solution containing a coloring substrate is added to the reaction vessel, and the enzyme-labeled antibody is colored by an enzymatic reaction.
[0009] 最後に、例えば水酸ィ匕ナトリウム水溶液などの溶菌抽出液によって、酵素標識化抗 体が結合した標的菌 102を溶出した後（図 12 (e) )、光源 105に対向して配置された 検出器 106において、色素が特異的に吸収する波長の光を検出し、抗原濃度を測 定する（図 12 (f) )。  [0009] Finally, the target bacterium 102 bound with the enzyme-labeled antibody is eluted with a lysis extract such as an aqueous sodium hydroxide solution (FIG. 12 (e)), and then placed opposite the light source 105. The detected detector 106 detects light having a wavelength that the dye specifically absorbs, and measures the antigen concentration (FIG. 12 (f)).
[0010] 以上説明したように、サンドイッチ法によれば、培養操作を必要とする検査と異なり 、煩雑な操作や専門的な知識がなくても適切な微生物検出を迅速に行うことができる [0011] 一方で、サンドイッチ法による測定操作よりも更に取り扱い易い検査キットを用いて[0010] As described above, according to the sandwich method, unlike a test requiring a culturing operation, appropriate microorganism detection can be rapidly performed without complicated operations or specialized knowledge. [0011] On the other hand, using an inspection kit that is easier to handle than the measurement operation by the sandwich method,
、微生物検出を迅速に行うものもある。例えば、アルカリホスファターゼと特異的に結 合して発色し得る発色基質成分を含有する基質液を展開可能な検出キットを用いる ことで、大腸菌の生菌のみを迅速に検出する技術が提案されている (特許文献 1参 照)。 In addition, there is also one that performs detection of microorganisms quickly. For example, a technique has been proposed for rapidly detecting only viable Escherichia coli by using a detection kit that can develop a substrate solution containing a chromogenic substrate component capable of specifically binding to alkaline phosphatase to develop color. (See Patent Document 1).
[0012] より具体的には、特許文献 1に記載の発明は、大腸菌と特異的に結合し得る特異 的結合成分を固定ィ匕した固定相を吸水性基材の表面上の任意の領域に形成してな る検出具に、被検液を展開させる工程と、被検液を展開した後の吸水性基材に、ァ ルカリホスファターゼと特異的に結合して発色し得る発色基質成分を含有する基質 液を展開させる工程と、を少なくとも有する検出方法及び検出キットである。  [0012] More specifically, the invention described in Patent Document 1 discloses a method in which a stationary phase in which a specific binding component capable of specifically binding to Escherichia coli is immobilized is applied to an arbitrary region on the surface of the water-absorbing substrate. The step of spreading the test solution on the formed detection device, and the step of developing the test solution containing a color-developing substrate component capable of specifically binding to alkaline phosphatase and coloring the water-absorbing substrate. And a detection kit comprising at least a step of developing a substrate solution.
[0013] この検出方法及び検出キットによれば、吸水性基材の吸水性を適宜調節することで 、被検液と基質液を展開させるスピードを最適なものとすることができ、ひいては検査 の迅速ィ匕を図ることができる。また、被検液中に大腸菌の生菌が存在する場合には、 特異的結合成分によって固定相に捕捉された生菌が有するアルカリホスファターゼ に対し、発色基質成分が特異的に結合して発色することから、被検液中における大 腸菌の生菌の有無まで検出することができる、というメリットがある。  [0013] According to the detection method and the detection kit, by appropriately adjusting the water absorption of the water-absorbent substrate, the speed at which the test liquid and the substrate liquid are developed can be optimized, and thus, the inspection speed can be improved. It is possible to make a quick dagger. When live Escherichia coli is present in the test solution, the chromogenic substrate component specifically binds to the alkaline phosphatase of the live bacteria captured on the stationary phase by the specific binding component to form a color. Therefore, there is an advantage that it is possible to detect even the presence or absence of viable E. coli in the test solution.
[0014] 特許文献 1 :特開 2002— 165599 (段落番号 [0033ト [0037])  Patent Document 1: Japanese Patent Application Laid-Open No. 2002-165599 (paragraph number [0033] [0037])
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0015] し力しながら、上述したサンドイッチ法及び特許文献 1記載の検出方法では、以下 のような問題が生ずる。 [0015] However, the sandwich method described above and the detection method described in Patent Document 1 have the following problems.
[0016] まず、サンドイッチ法では、抗原の検出までに 2回の抗原抗体反応（図 12 (b) , (c) 参照）が必要となり、各回の抗原抗体反応ともに一定時間を要してしまうことから、検 查の更なる迅速ィ匕を図ることができないといった問題がある。また、サンドイッチ法で は、一般的に、大腸菌の外膜に存在するリポ多糖に特異的に結合する抗体を用いる ことから、大腸菌が生菌であるか、或いは死菌ゃ菌断片であるか、を区別することが できず、死菌ゃ菌断片のみを含み食中毒被害をもたらさない適合品を、食品等の検 查段階で排除してしまうといった問題もある。 [0017] また、特許文献 1記載の検出方法では、上述のとおり、検査の迅速化及び大腸菌 の生菌の検出は可能になるものの、 0. Olml— 0. 2mlというごく少量の試料しか扱え ないことから (特許文献 1明細書段落番号 [0034]参照）、検査の確実性を担保する ことができないといった問題がある。すなわち、検査対象となる試料が少量である場 合には、大腸菌の含有確率の低下とともに、大腸菌のうちの生菌含有確率の低下も 招いてしまうため、必然的に、検出精度 ·検出感度が低くなつてしまう。 [0016] First, in the sandwich method, two antigen-antibody reactions (see Figs. 12 (b) and 12 (c)) are required until antigen detection, and each antigen-antibody reaction requires a certain amount of time. Therefore, there is a problem that it is not possible to achieve further rapid inspection. In addition, since the sandwich method generally uses an antibody that specifically binds to lipopolysaccharide present on the outer membrane of Escherichia coli, whether the Escherichia coli is a viable cell or a dead bacterial cell fragment is used. There is also a problem in that conformable products that contain only dead bacteria and bacterial fragments and do not cause food poisoning are excluded during the inspection of foods and the like. [0017] Further, as described above, the detection method described in Patent Document 1 can speed up the test and detect live bacteria of Escherichia coli, but can handle only a very small amount of sample of 0.2 Olml to 0.2 ml. Therefore, there is a problem that the reliability of the inspection cannot be ensured (see Paragraph No. [0034] of Patent Document 1). In other words, when the amount of the sample to be tested is small, the probability of containing E. coli and the probability of containing viable bacteria in E. coli also decrease. It will be low.
[0018] 特に、食品工場で食中毒が発覚した場合でも、流通経路によってはすでに小売店 で販売され、消費者の口の中に入って力 対応するケースが多ぐ食中毒被害が広 力 ¾危険性が高ぐ検査の更なる迅速ィ匕が望まれていた。  [0018] In particular, even when food poisoning is discovered in a food factory, it is already sold at retail stores depending on the distribution channel, and there are many cases in which it enters the mouth of consumers and responds strongly. There has been a demand for an even faster inspection with a high level of inspection.
[0019] 本発明は以上の点に鑑みてなされたものであり、その目的は、抗原としての微生物 のうちの生菌を短時間で迅速に検出することができ、加えて検査の確実性をも担保し 得る検出方法及び検出装置を提供することにある。  [0019] The present invention has been made in view of the above points, and an object of the present invention is to enable rapid detection of viable bacteria among microorganisms serving as antigens in a short time, and to increase the reliability of the test. Another object of the present invention is to provide a detection method and a detection device that can secure the same.
課題を解決するための手段  Means for solving the problem
[0020] 以上のような課題を解決するために、本発明は、大腸菌などの被検抗原に、その被 検抗原内の生菌によって酵素分解される標識化物質を作用させた標識化抗原を生 成した後、被検抗原に特異的に結合し得る特異的結合抗体を固定ィ匕してなる固定 相にお 、て、その標識ィ匕抗原を捕捉することを特徴とする。 [0020] In order to solve the above-described problems, the present invention provides a test antigen such as Escherichia coli with a labeled antigen obtained by allowing a labeling substance which is enzymatically decomposed by viable bacteria in the test antigen to act on the antigen. After generation, the labeled antigen is captured in a stationary phase in which a specific binding antibody capable of specifically binding to the test antigen is immobilized.
[0021] より具体的には、本発明は、以下のものを提供する。 [0021] More specifically, the present invention provides the following.
[0022] (1) 被検抗原と、前記被検抗原内の生菌によって酵素分解される標識化物質と、 の作用によって、前記被検抗原内の生菌を特異的に標識化して検出する検出方法 であって、前記被検抗原に前記標識化物質を作用させて光学的に検出可能な標識 化抗原を生成し、前記被検抗原に特異的に結合しうる特異的結合抗体を固定化して なる固定相において、前記標識化抗原を捕捉することを特徴とする検出方法。  [0022] (1) The action of the test antigen and a labeling substance which is enzymatically decomposed by the live bacteria in the test antigen, specifically labels and detects the live bacteria in the test antigen. A detection method, wherein the labeled substance is allowed to act on the test antigen to generate an optically detectable labeled antigen, and a specific binding antibody capable of specifically binding to the test antigen is immobilized. A detection method comprising capturing the labeled antigen in a stationary phase.
[0023] 本発明によれば、大腸菌などの被検抗原 (生菌'死菌を含む）と、その被検抗原のう ちの生菌によって酵素分解される標識化物質と、の作用によって生菌を検出する検 出方法であって、被検抗原に標識ィ匕物質を作用させて蛍光反応等によって光学的 に検出可能な標識化抗原を生成し、抗原抗体反応によって被検抗原に特異的に結 合しうる特異的結合抗体を固定ィ匕してなる固定相において、その標識ィ匕抗原を捕捉 することとしたから、捕捉対象は、光学的に検出可能な標識化抗原 (生菌)と、標識ィ匕 されておらず光学的に検出不可能な死菌 (菌断片）とになる。 According to the present invention, the activity of a test antigen (including dead bacteria) such as Escherichia coli and a labeled substance which is enzymatically degraded by the live bacteria among the test antigens is evaluated. This is a detection method for detecting labeled antigens, in which a labeled antigen is allowed to act on a test antigen to generate a labeled antigen that can be optically detected by a fluorescence reaction or the like, and the antigen-antibody reaction specifically reacts with the test antigen. In a stationary phase formed by immobilizing a specific binding antibody capable of binding, the labeled antibody is captured. Therefore, the capture targets are an optically detectable labeled antigen (live bacteria) and a dead bacterium (fungal fragment) that is not labeled and cannot be detected optically.
[0024] 従って、従来のサンドイッチ法では必須の二次抗体が不要となり、その結果、従来 は 2回必要であった抗原抗体反応が 1回で済むこととなり、ひいては検査の迅速ィ匕を 図ることができる。また、捕捉した被検抗原のうち、光学的に検出可能な抗原は、標 識ィ匕抗原としての生菌のみであることから、生菌 Z死菌を区別して検出することが可 能となり、食中毒被害をもたらす生菌を的確に検出することができる。  [0024] Therefore, the conventional sandwich method does not require an essential secondary antibody, and as a result, the antigen-antibody reaction, which was required twice in the past, can be performed only once. Can be. In addition, among the captured test antigens, the only optically detectable antigen is a live bacterium serving as the labeling antigen, so that it is possible to detect live bacteria Z and dead bacteria separately. Viable bacteria causing food poisoning damage can be accurately detected.
[0025] さらに、従来の特許文献 1記載の検出方法で扱う試料の量 (0. Olml— 0. 2ml)と 比較して、本発明に係る検出方法で扱う試料の量は、数十 ml—数百 mlであることか ら、サンプル抽出に起因した被検抗原の含有確率の低下を防ぐことができ、ひいて は検出精度 ·検出感度の低下を防ぎ、検査の確実性を担保することができる。  [0025] Furthermore, compared to the amount of sample (0.2 Olml-0.2 ml) handled by the conventional detection method described in Patent Document 1, the amount of sample handled by the detection method according to the present invention is several tens ml- Since the sample volume is several hundred ml, it is possible to prevent a decrease in the probability of containing the test antigen due to sample extraction, thereby preventing a decrease in detection accuracy and detection sensitivity and ensuring test reliability. it can.
[0026] (2) 前記固定相において、増殖培養液によって増殖した前記標識化抗原を捕捉 することを特徴とする検出方法。  (2) A detection method comprising capturing the labeled antigen grown by a growth medium in the stationary phase.
[0027] 本発明によれば、上述した固定相において、増殖培養液によって増殖した標識ィ匕 抗原を捕捉することしたので、増殖培養液を添加する前と比べて標識化抗原濃度を 高めることができ、その結果、標識ィ匕抗原の捕捉確率が向上し、ひいては検出精度' 検出感度を上げることができる。  According to the present invention, in the stationary phase described above, the labeled antigen grown in the growth medium is captured, so that the concentration of the labeled antigen can be increased as compared to before the addition of the growth medium. As a result, the probability of capturing the labeled antigen can be improved, and the detection accuracy and the detection sensitivity can be increased.
[0028] (3) 前記標識化抗原を含む試料溶液を複数回循環させながら、前記固定相にお いて、循環される標識化抗原を捕捉することを特徴とする検出方法。  (3) A detection method comprising capturing the circulated labeled antigen in the stationary phase while circulating the sample solution containing the labeled antigen a plurality of times.
[0029] 本発明によれば、上述した標識化抗原を含む試料溶液を複数回循環させながら、 上述した固定相において、循環される標識ィ匕抗原を捕捉することとしたから、一次抗 体が固定化された固定相に試料溶液を複数回接触させることができ、標識ィ匕抗原の 捕捉確率が向上し、検出精度'検出感度を向上することができる。  According to the present invention, the circulating labeled antigen is captured in the stationary phase while the sample solution containing the labeled antigen is circulated a plurality of times. The sample solution can be brought into contact with the immobilized stationary phase a plurality of times, so that the probability of capturing the labeled antigen can be improved, and the detection accuracy and the detection sensitivity can be improved.
[0030] (4) 複数種類の前記被検抗原のそれぞれに対応して特異的に結合しうる特異的 結合抗体を固定ィ匕してなる複数種類の固定相において、前記被検抗原を捕捉する ことを特徴とする検出方法。  (4) The test antigen is captured in a plurality of stationary phases formed by immobilizing a specific binding antibody capable of specifically binding to each of the plurality of test antigens. A detection method characterized in that:
[0031] 本発明によれば、複数種類の上述した被検抗原のそれぞれに対応して特異的に 結合しうる特異的結合抗体を固定ィ匕してなる複数種類の固定相において、複数種類 の被検抗原を一連の検査フローの中で 1度にまとめて捕捉することとしたから、検査 の迅速化 ·効率ィ匕を図ることができる。 According to the present invention, in a plurality of types of stationary phases formed by immobilizing a specific binding antibody capable of specifically binding to each of a plurality of types of test antigens described above, Since the test antigens are collectively captured at one time in a series of test flows, the test can be speeded up and efficiency can be improved.
[0032] (5) 被検抗原に特異的に結合しうる特異的結合抗体を固体ィ匕してなる固定相を 設置可能なカラムを有する検出装置であって、前記固定相が設置されたカラムにお V、て、前記被検抗原が標識化された標識化抗原を捕捉することを特徴とする検出装 置。  (5) A detection device having a column capable of installing a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen, wherein the column is provided with the stationary phase. 5. A detection device, wherein the test antigen captures a labeled antigen labeled.
[0033] 本発明によれば、大腸菌などの被検抗原 (生菌'死菌を含む）に特異的に結合しう る特異的結合体を固定ィ匕してなる固定相を設置可能なカラム (バイオカラム)を有す る検出装置で、上述した固定相が設置されたカラムにおいて、生菌のみに作用する 標識ィ匕物質によって被検抗原が標識化された標識ィ匕抗原を捕捉することとしたから、 標的化抗原捕捉までに 1回の抗原抗体反応で足りることなり、検査の迅速ィ匕を図ると ともに、食中毒被害をもたらす生菌を的確に検出することができる。また、大量の試料 を一気に扱うことができることから、サンプル抽出に起因した被検抗原の含有確率の 低下を防ぐことができ、ひいては検出精度 ·検出感度の低下を防ぎ、検査の確実性を 担保することができる。  According to the present invention, a column in which a stationary phase formed by immobilizing a specific conjugate capable of specifically binding to a test antigen (including live bacteria and dead bacteria) such as Escherichia coli can be installed. In a detection device having a (bio column), a column on which the stationary phase described above is installed captures labeled antigens in which the test antigen is labeled by the labeled antigens acting only on viable bacteria. Thus, a single antigen-antibody reaction is sufficient before capturing the targeted antigen, which enables rapid testing and enables accurate detection of viable bacteria causing food poisoning damage. In addition, since a large number of samples can be handled at once, it is possible to prevent a decrease in the probability of containing the test antigen due to sample extraction, thereby preventing a decrease in detection accuracy and detection sensitivity, and ensuring test reliability. be able to.
[0034] (6) 前記検出装置は、さらに、液体を攪拌する攪拌装置を備え、前記標識化抗原 は、前記攪拌装置において標識化されることを特徴とする検出装置。  (6) The detection device, further comprising a stirring device for stirring the liquid, wherein the labeled antigen is labeled by the stirring device.
[0035] 本発明によれば、上述した検出装置は、さらに、液体 (試料溶液)を機械的に攪拌 する攪拌装置を備えており、上述した標識ィ匕抗原は、この攪拌装置において標識ィ匕 されることとしたから、被検抗原の標識化を促進し、ひいては標識化抗原を効率的に 生成することができる。  According to the present invention, the above-described detection device further includes a stirring device for mechanically stirring the liquid (sample solution), and the above-described labeled antigen is added to the labeling antigen in the stirring device. Therefore, the labeling of the test antigen can be promoted, and the labeled antigen can be efficiently produced.
[0036] (7) 前記カラムは、複数回使用可能であることを特徴とする検出装置。  (7) The detection device, wherein the column can be used a plurality of times.
[0037] 本発明によれば、上述したカラムは、複数回使用可能であることから、複数回の微 生物検査を連続して効率的に行うことができる。  According to the present invention, since the above-mentioned column can be used a plurality of times, a plurality of microbiological tests can be continuously and efficiently performed.
[0038] (8) 被検抗原に特異的に結合しうる特異的結合抗体を固体ィ匕してなる固定相を 設置可能なカラムを複数有する検出装置であって、前記固定相が設置された複数の カラムにお ヽて、前記被検抗原が標識化された標識化抗原を捕捉することを特徴と する検出装置。 [0039] 本発明によれば、被検抗原に特異的に結合しうる特異的結合抗体を固体化してな る固定相を設置可能なカラムを複数 (複数種類)有する検出装置であって、その固定 相が設置された複数のカラムにぉ ヽて、被検抗原が標識化された標識化抗原を捕 捉することとしたから、検査の迅速化 ·効率ィ匕を図ることができる。 (8) A detection device having a plurality of columns capable of installing a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen, wherein the stationary phase is installed. A detection apparatus characterized in that the test antigen captures a labeled antigen in a plurality of columns. According to the present invention, there is provided a detection device having a plurality (a plurality of types) of columns on which a stationary phase formed by solidifying a specific binding antibody capable of binding specifically to a test antigen can be provided. Since the labeled antigen in which the test antigen is labeled is captured by using a plurality of columns provided with a stationary phase, the test can be performed quickly and efficiently.
[0040] (9) 被検抗原に特異的に結合しうる特異的結合抗体を固定化してなる固定相が 設置されたバイオカラム。  (9) A biocolumn provided with a stationary phase in which a specific binding antibody capable of specifically binding to a test antigen is immobilized.
[0041] 本発明によれば、被検抗原に特異的に結合しうる特異的結合抗体を固定ィ匕してな る固定相が設置されたバイオカラムを提供することで、より検査の迅速化'確実ィ匕を 図ることができる。なお、例えば凍結乾燥法などの保存手段を用いることで、このバイ ォカラムを長期間安定ィ匕状態で保存することができる。  According to the present invention, by providing a biocolumn provided with a stationary phase obtained by immobilizing a specific binding antibody capable of specifically binding to a test antigen, the test can be further speeded up. 'I can make sure. By using a storage means such as a freeze-drying method, the biocolumn can be stored in a stable state for a long time.
[0042] (10) 被検抗原に特異的に結合しうる特異的結合抗体を固定化してなる固定相が 設置されたバイオカラムにおいて、前記バイオカラム内の圧力変動を利用して、前記 固定相を攪拌する方法。  (10) In a biocolumn provided with a stationary phase in which a specific binding antibody capable of specifically binding to a test antigen is immobilized, pressure fluctuation in the biocolumn is used to make use of the stationary phase. How to stir.
[0043] 本発明によれば、バイオカラムに急激な圧力変動を生じさせてバイオカラム内を流 れる検水の流速が増す結果、バイオカラム内で固定相が攪拌されることになるので、 固定相と循環する試料溶液 (検水）とを十分に接触させることができ、高い検出精度 ,検出感度を維持することができる。  According to the present invention, abrupt pressure fluctuations occur in the biocolumn to increase the flow rate of the test water flowing in the biocolumn, so that the stationary phase is agitated in the biocolumn. The phase and the circulating sample solution (sample) can be brought into sufficient contact, and high detection accuracy and detection sensitivity can be maintained.
発明の効果  The invention's effect
[0044] 以上説明したように、本発明は、従来は 2回必要であった抗原抗体反応を 1回で済 むようにしたものであることから検査の迅速ィ匕を図ることができ、また、光学的に検出 する抗原を標識化抗原 (生菌）とするものであることから生菌 Z死菌の判別が可能に なり、さらに、大量の試料を扱えることから検査の確実性を担保することができるもの である。  As described above, according to the present invention, the antigen-antibody reaction, which was conventionally required twice, can be performed only once, so that the test can be performed quickly. Since the antigens to be detected are labeled antigens (live bacteria), it is possible to distinguish between live bacteria and dead bacteria.Moreover, since a large number of samples can be handled, the reliability of the test can be ensured. You can do it.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0045] 以下、本発明を実施するための最良の形態について、図面に基づいて説明する。 Hereinafter, the best mode for carrying out the present invention will be described with reference to the drawings.
[0046] [検出装置] [Detection device]
図 1は、本発明の実施の形態に係る検出装置 1の外観図である。  FIG. 1 is an external view of a detection device 1 according to an embodiment of the present invention.
[0047] 図 1において、本発明の実施の形態に係る検出装置 1は、内部にポンプやバルブ を有する箱状のコントロールボックスの側面に、標的菌を捕捉する固定相が設置され たバイオカラム 2を有している。また、そのノ ィオカラム 2の隣には、捕捉後の標的菌 を溶出する溶菌液が入った M— Cell3が設置され、検出装置 1の上面には、例えばボ トル B1— B5の 5個のボトル (数は問わない）が設置されている。 In FIG. 1, a detection device 1 according to an embodiment of the present invention has a pump and a valve inside. A biocolumn 2 provided with a stationary phase for capturing a target bacterium is provided on a side surface of a box-shaped control box having the following. An M-Cell 3 containing a lysate that elutes the captured target bacterium is installed next to the No. 2 column. On the upper surface of the detector 1, for example, five bottles B1 to B5 are placed. (Any number) is set up.
[0048] ここで、図 2は、本発明の実施の形態に係る検出 1に設置されたバイオカラム 2の拡 大図である。図 2において、このバイオカラム 2は、バイオカラム用ガラス管の内部に、 抗原抗体反応によって抗原を捕捉し得るガラスビーズを充填することによって作製さ れる。 Here, FIG. 2 is an enlarged view of the biocolumn 2 installed in the detection 1 according to the embodiment of the present invention. In FIG. 2, the biocolumn 2 is prepared by filling glass beads for a biocolumn into glass beads capable of capturing an antigen by an antigen-antibody reaction.
[0049] より具体的には、まず、水酸ィ匕ナトリウム水溶液や塩酸によってガラスビーズを前処 理した後、一晩これを乾燥させる。そして、このガラスビーズに、焼結処理、シリルイ匕 剤によるシリルイ匕処理を施した後、リンスを行 、室温で乾燥させることによってシリル 化ガラスビーズを作製する。  More specifically, first, the glass beads are pre-treated with an aqueous sodium hydroxide solution or hydrochloric acid, and then dried overnight. Then, the glass beads are subjected to a sintering treatment and a silyllizing treatment with a silylating agent, rinsed, and dried at room temperature to produce silylated glass beads.
[0050] 次いで、このシリル化ガラスビーズをバイオカラム用ガラス管の中に約 0. 5グラム充 填する。そして、カップリング剤としてのダルタルアルデヒドを含むダルタルアルデヒド 溶液に数十分浸漬し、リン酸緩衝液で洗浄した後、非特異的吸着によって一次抗体 固定ィ匕処理を行う。この一次抗体固定ィ匕処理では、適宜バイオカラム用ガラス管を 洗浄することで、未反応であった一次抗体を除去する。  [0050] Next, about 0.5 g of the silylated glass beads is filled in a glass tube for a biocolumn. Then, it is immersed for several minutes in a dartartaldehyde solution containing dartartaldehyde as a coupling agent, washed with a phosphate buffer solution, and then subjected to a primary antibody immobilization treatment by non-specific adsorption. In the primary antibody immobilization treatment, the unreacted primary antibody is removed by appropriately washing the glass tube for the biocolumn.
[0051] 次いで、一次抗体固定ィ匕処理が完了したら、ブロッキング剤としてのゥシアルブミン 血清を含むブロッキング溶液を注入し、シリルイ匕ガラスビーズの表面に残存する非特 異的吸着面にブロッキング剤を非特異的吸着させ、それ以後、他の有機物質などの 非特異的吸着が起こるのを防止する。  [0051] Next, when the primary antibody immobilization treatment is completed, a blocking solution containing sialic albumin serum as a blocking agent is injected, and the blocking agent is non-specifically applied to the non-specific adsorption surface remaining on the surface of the glass beads. Adsorb and prevent subsequent non-specific adsorption of other organic substances.
[0052] 最後に、リン酸緩衝液などでバイオカラム用ガラス管内を複数回洗浄して、未反応 であったブロッキング剤を除去することによってノィォカラム 2が作製される。なお、例 えば凍結乾燥法などの保存手段を用いることで、このバイオカラム 2を長期間安定ィ匕 状態で保存することができる。  [0052] Finally, the inside of the glass tube for the biocolumn is washed a plurality of times with a phosphate buffer or the like to remove the unreacted blocking agent, whereby the Noocolumn 2 is produced. By using a storage means such as a freeze-drying method, for example, the biocolumn 2 can be stored in a stable state for a long time.
[0053] なお、本発明の実施の形態ではガラスビーズを用いたバイオカラム 2の作製にっ ヽ て説明したが、本発明はこれに限られることなぐ例えば、シリル化処理やカップリン グ処理などの各種条件を確立するために比較的扱 、の容易な平面系ガラスを用いる こととしてもよい。また、本発明の実施の形態では球状のガラスビーズを用いているが 、ガラスビーズは抗体を固定ィヒする担体 (抗体を固定ィヒする母体)の一つであり、抗 体を固定ィ匕できるだけの表面積を有し、それをカラムに充填した状態で抗体と検水 が十分に接触することができる形態の担体であれば、 、かなる形状のものであっても よい。作製工程については、上述したガラスビーズを用いたノィォカラム 2の作製ェ 程とほぼ同じであるので、その説明を省略する。また、本発明は、固定化層表面に一 次抗体が固定できれば、ビーズの材質、シリル化処理条件、一次抗体固定化方法の 如何を問わず適用可能である。 Although the embodiment of the present invention has been described with respect to the production of the biocolumn 2 using glass beads, the present invention is not limited to this. For example, a silylation treatment, a coupling treatment, etc. Uses relatively easy-to-handle flat glass to establish various conditions It may be good. Further, in the embodiment of the present invention, spherical glass beads are used. However, glass beads are one of carriers for immobilizing antibodies (a matrix for immobilizing antibodies), and are used for immobilizing antibodies. The carrier may have any shape as long as the carrier has a surface area as much as possible and the antibody and the sample can be sufficiently brought into contact with the sample in a state where the column is packed. The production process is almost the same as the production process of the Noo column 2 using the glass beads described above, and thus the description thereof is omitted. In addition, the present invention is applicable irrespective of the material of the beads, the silylation treatment conditions, and the method of immobilizing the primary antibody, as long as the primary antibody can be immobilized on the surface of the immobilization layer.
[0054] [検査工程] [Inspection process]
図 3は、図 1に示す検出装置 1を用いて微生物検査を行う際の流路系統図である。 また、図 4は、図 3に示す流路系統図における検査工程の概略を示すフローチャート である。  FIG. 3 is a flow path system diagram when performing a microorganism test using the detection device 1 shown in FIG. FIG. 4 is a flowchart schematically showing an inspection process in the flow path diagram shown in FIG.
[0055] なお、図 3において、ボトル B1には、染色薬として 6—カルボキシルフルォレセインジアセテート、希釈 (CFDA希釈)液 (標識ィ匕物質を含む溶液)を添加し、加水分解 によって標識化された生菌 (標識化抗原)と、加水分解が行われなカゝつた死菌とが混 在する被検抗原を含む検水（例えば 100ml)が注入されており、ボトル B5及びボトル B6には、カラム洗净液としてのリン酸緩衝液が注入されており、 M— Cell3には、バイ ォカラム 2において捕捉された標的菌を溶出するアルカリ水溶液が注入されている。 また、本実施形態で用いた CFDAは、生菌を染色することができ、かつ、それらを蛍 光発色などで検出できる薬剤で代替可能である。  In FIG. 3, 6-carboxylfluorescein diacetate, a dilution (CFDA dilution) solution (solution containing a labeling substance) was added to bottle B1 as a staining agent, and labeled by hydrolysis. A test sample (for example, 100 ml) containing a test antigen containing a mixture of live bacteria (labeled antigen) and dead dead bacteria that have not been hydrolyzed is injected into bottles B5 and B6. In this example, a phosphate buffer solution as a column washing solution is injected, and an alkaline aqueous solution that elutes target bacteria captured in the biocolumn 2 is injected into M-Cell3. Further, CFDA used in the present embodiment can be used as an agent capable of staining viable bacteria and detecting them by fluorescence or the like.
[0056] 図 4において、まず、固定ィ匕工程が実行される (ステップ Sl)。より具体的には、図 3 において、ポンプ P1を作動させることによって、ボトル B1に注入された CFDA希釈 液の添加検水が、ボトル Bl→バルブ Vl→バルブ V2→ポンプ Pl→バルブ 3→バ ィォカラム 2→バルブ 4→バルブ 5→バルブ 6→ボトル B2の順序で流動する。所要時 間はおよそ 15分である。そして、バルブ VIを切り替えることで、ボトル B2に貯留して Vヽる検水が、ボトル B2→バルブ Vl→バルブ V2→ポンプ Pl→バルブ V3→バイオ力 ラム 2→バルブ 4→バルブ 5→バルブ 6→ボトル B3の順序で流動する。この所要時間 もおよそ 15分である。以上ステップ S1の固定ィ匕工程によって、バイオカラム 2 (ガラス ビーズ)に固定化された一次抗体に、検水中の被検抗原を抗原抗体反応により特異 的に結合させる。なお、ボトル B1に増殖培養液を添加しておき、この増殖培養液によ つて増殖した標識ィ匕抗原を捕捉することとしてもよい。これにより、標識化抗原濃度を 高めることができ、標識ィ匕抗原の捕捉確率を向上することができる。 In FIG. 4, first, the fixing step is performed (Step Sl). More specifically, in Fig. 3, by operating the pump P1, the addition test of the CFDA diluent injected into the bottle B1 changes the bottle Bl → valve Vl → valve V2 → pump Pl → valve 3 → biocolumn. Flows in the order of 2 → valve 4 → valve 5 → valve 6 → bottle B2. The required time is about 15 minutes. Then, by switching valve VI, the sampled water stored in bottle B2 and flowing into the bottle B2 is detected as bottle B2 → valve Vl → valve V2 → pump Pl → valve V3 → biopower ram 2 → valve 4 → valve 5 → valve 6 → Flow in the order of bottle B3. This takes about 15 minutes. As described above, the biocolumn 2 (glass The test antigen in the sample is specifically bound to the primary antibody immobilized on the beads by an antigen-antibody reaction. Note that a growth culture solution may be added to the bottle B1, and the labeled antigen grown by the growth culture solution may be captured. Thereby, the concentration of the labeled antigen can be increased, and the probability of capturing the labeled antigen can be improved.
[0057] ここで、高い検出精度，検出感度を維持するためには、特異的結合抗体を固定ィ匕 してなる固定相 (ガラスビーズ)と、循環する試料溶液 (検水）とを十分に接触させなけ ればならない。そこで、本実施形態では、電磁式ピンチバルブ PV (図 3参照）を利用 して、ノィォカラム 2内のガラスビーズ（固定相）を効果的に攪拌するようにして!/、る。 より具体的には、図 5を用いて説明する。図 5は、固定相を効果的に攪拌する様子を 示す説明図である。 Here, in order to maintain high detection accuracy and detection sensitivity, the stationary phase (glass beads) in which the specific binding antibody is immobilized and the circulating sample solution (sample) are sufficiently used. They must be in contact. Therefore, in this embodiment, the glass beads (stationary phase) in the Noo column 2 are effectively agitated using an electromagnetic pinch valve PV (see FIG. 3)! More specifically, this will be described with reference to FIG. FIG. 5 is an explanatory diagram showing how the stationary phase is effectively stirred.
[0058] 図 5において、バイオカラム 2とバルブ V3との間に設けられたピンチバルブ PVが、 開状態 (ON状態)から閉状態 (OFF状態）になると（図 5左図→図 5中央図）、ピンチ バルブ PV力もバイオカラム 2へは検水が流動しなくなるとともに、ピンチバルブ PVの バルブ V3側の配管圧力が増加する。そして、所定時間経過後、ピンチバルブ PVが 閉状態 (OFF状態)から開状態 (ON状態）になると（図 5中央図→図 5右図）、ピンチ バルブ PV力 バイオカラム 2へ再び検水が流動する。  In FIG. 5, when the pinch valve PV provided between the biocolumn 2 and the valve V3 changes from an open state (ON state) to a closed state (OFF state) (FIG. 5, left view → FIG. 5, center view) ), The pinch valve PV force also stops the test water from flowing to the biocolumn 2, and the piping pressure on the valve V3 side of the pinch valve PV increases. After a predetermined time, when the pinch valve PV changes from the closed state (OFF state) to the open state (ON state) (Fig. 5 center view → Fig. 5 right view), water is again detected in the biocolumn 2 of the pinch valve PV force. Flow.
[0059] このとき、ピンチバルブ PVを所定時間閉状態 (OFF状態）にしていたことに起因し て、バイオカラム 2内では急激な圧力変動が生じ、バイオカラム 2内を流れる検水の 流速が増す。その結果、バイオカラム 2内でガラスビーズ（固定相）が攪拌されること になる（図 5右図参照)。  [0059] At this time, due to the pinch valve PV being closed (OFF state) for a predetermined time, a sharp pressure fluctuation occurs in the biocolumn 2 and the flow rate of the test water flowing through the biocolumn 2 decreases. Increase. As a result, the glass beads (stationary phase) are stirred in the biocolumn 2 (see the right figure in Fig. 5).
[0060] このように、本実施形態では、検水をバイオカラム 2に循環させる際に、ピンチバル ブ PVを所定のタイミングで開閉することによって、固定相（ガラスビーズ)が定期的か つ効果的に攪拌されるような構成としている。これ〖こより、固定相 (ガラスビーズ)と検 水とを十分に接触させることが可能となって 、る。  As described above, in the present embodiment, when the test sample is circulated through the biocolumn 2, the pinch valve PV is opened and closed at a predetermined timing, so that the stationary phase (glass beads) is periodically and effectively. It is configured to be stirred. This makes it possible to bring the stationary phase (glass beads) into contact with the sample sufficiently.
[0061] なお、本実施形態では、電磁式のピンチバルブ PVを利用することとしたが、本発明 はこれに限られず、例えば、手動式 ·電動式のピンチバルブを利用することとしてもよ い。また、バイオカラム 2内の固定相が適度に攪拌される効果を奏する手段であれば 如何なるものを利用してもよぐ特に、ピンチバルブに限定されない。 [0062] 次いで、洗浄工程が実行される (ステップ S2)。より具体的には、図 3において、ノ ルブ V2を切り替えることで、ボトル B5に貯留しているリン酸緩衝液力 ボトル 5→バ ルブ 2→ポンプ Pl→バルブ V3→バイオカラム 2→バルブ V4→バルブ V5→ボトル 4 の順序で流動する。その後、ポンプ P1のスィッチを切る。所要時間はおよそ 15分で ある。以上ステップ S2の洗浄工程によって、リン酸緩衝液をバイオカラム 2に流し、未 反応であった一次抗体などを洗浄し、結果として被検抗原の凝縮化を行う。 [0061] In the present embodiment, the electromagnetic pinch valve PV is used. However, the present invention is not limited to this. For example, a manual or electric pinch valve may be used. . In addition, any means may be used as long as the stationary phase in the biocolumn 2 has an effect of being appropriately stirred, and is not particularly limited to a pinch valve. Next, a cleaning step is performed (Step S2). More specifically, in Figure 3, by switching the valve V2, the phosphate buffer power stored in bottle B5 bottle 5 → valve 2 → pump Pl → valve V3 → biocolumn 2 → valve V4 → Flow in the order of valve V5 → bottle 4. Then, switch off the pump P1. It takes about 15 minutes. By the washing step in step S2, the phosphate buffer is passed through the biocolumn 2 to wash unreacted primary antibodies and the like, and condensate the test antigen as a result.
[0063] 次いで、溶出工程が実行される (ステップ S3)。より具体的には、図 3において、バ ルブ V3及びバルブ V4を切り替え、被検抗原を溶出する溶菌液が入った M— Cell3 及びポンプ P2を作動させることによって、バイオカラム 2において捕捉された被検抗 原を溶出する。そして、 Flow Cell (図 3中の右下）を備えた蛍光分光光度計におい て、被検抗原のうち標識化された生菌 (標識化抗原)を光学的に検出 (スペクトル測 定)する。以上ステップ S3の溶出工程によって、食中毒の被害をもたらす生菌のみの 検出を行う。そして、以上ステップ S1—ステップ S3までの一連の工程によって、微生 物検査は一応終了する。  Next, an elution step is performed (Step S3). More specifically, in FIG. 3, by switching valve V3 and valve V4 and activating M-Cell3 containing the lysate for eluting the antigen to be tested and pump P2, the sample captured in biocolumn 2 was changed. Elute the test antigen. Then, a fluorescent spectrophotometer equipped with a flow cell (lower right in FIG. 3) optically detects (spectrum measurement) labeled live bacteria (labeled antigen) among the test antigens. Through the elution step of step S3, only viable bacteria causing food poisoning damage are detected. Then, the microbial examination is temporarily terminated by a series of steps from step S1 to step S3.
[0064] なお、ボトル B4やボトル B6に実験数回分の薬液を注入して連続的に微生物検査 を行う場合には、図 4に示すように、洗浄工程が追加実行される (ステップ S4)。より具 体的には、図 3において、ポンプ P2を作動させ、バルブ V4及びバルブ V7を切り替え 、ボトル B6に貯留しているリン酸緩衝液によってバイオカラム 2を洗浄する。  [0064] When a microbe test is continuously performed by injecting a chemical solution for several experiments into bottle B4 or bottle B6, a washing step is additionally performed as shown in Fig. 4 (step S4). More specifically, in FIG. 3, the pump P2 is operated, the valve V4 and the valve V7 are switched, and the biocolumn 2 is washed with the phosphate buffer stored in the bottle B6.
[0065] 以上説明したように、図 4に示すステップ S1—ステップ S3 (ステップ S4)の一連の検 查工程によれば、抗原としての微生物のうち生菌のみを検出可能であることが分かる 。また、本発明の実施の形態に係る検出装置 1で検査可能な検水の量は、検査キット 等で用いる検水の量 (約 0. Olml— 0. 2ml)とは異なり、数十 ml—数百 mlであること から、試料のサンプルィ匕に伴って生じる大腸菌の含有確率の低下を防ぐことができ、 ひいては検出精度 ·検出感度を向上させることができる。なお、洗浄、溶出、洗浄の 各工程で使用される薬品及びその方法については、本発明の趣旨を逸脱しない範 囲で変更可能である。  As described above, according to the series of detection steps from Step S1 to Step S3 (Step S4) shown in FIG. 4, it can be seen that only viable bacteria can be detected from microorganisms as antigens. In addition, the amount of water that can be tested by the detection device 1 according to the embodiment of the present invention is different from the amount of water to be used in a test kit or the like (about 0.2 ml—0.2 ml), and several tens ml— Since it is several hundred ml, it is possible to prevent a decrease in the probability of containing Escherichia coli caused by sample siding of the sample, and to improve detection accuracy and detection sensitivity. The chemicals used in each of the steps of washing, elution, and washing and the method thereof can be changed without departing from the spirit of the present invention.
[0066] さらに、図 4に示すステップ S1—ステップ S3 (ステップ S4)の一連の検査工程によ れば、従来のサンドイッチ法よりも短時間で微生物検査を行うことができる。検出装置 1を用いた検査の迅速ィ匕にっ 、て、図 6の模式図を用いて以下に詳述する。 Further, according to the series of inspection steps from step S1 to step S3 (step S4) shown in FIG. 4, the microorganism test can be performed in a shorter time than the conventional sandwich method. Detector In the following, a detailed description will be given of a quick test using the method 1 with reference to the schematic diagram of FIG.
[0067] [模式図] [0067] [Schematic diagram]
図 6は、本発明の実施の形態に係る検出方法の主要工程を示す模式図である。 (a )は抗体 (一次抗体)の固定ィ匕工程であり、（b)は被検抗原を含む試料液と蛍光試薬 の攪拌工程であり、（c)は標識ィ匕抗原を含む被検抗原の捕捉工程であり、（d)は被 検抗原の固定ィ匕工程であり、（e)は被検抗原の溶出工程であり、（f)は溶出させた被 検抗原のうち、標識ィ匕抗原のみを検出する検出工程である。なお、図 6 (a)—（f)に は、固定化層表面 10、一次抗体 11、標的菌 (生菌） 12、標識化物質 13、標識化抗 原 14、光源 15、検出器 16が記載されている。  FIG. 6 is a schematic diagram showing main steps of the detection method according to the embodiment of the present invention. (a) is the step of immobilizing the antibody (primary antibody), (b) is the step of stirring the sample solution containing the test antigen and the fluorescent reagent, and (c) is the test antigen containing the labeled antibody. (D) is the step of immobilizing the test antigen, (e) is the step of eluting the test antigen, and (f) is the step of immobilizing the labeled antigen among the eluted test antigens. This is a detection step for detecting only the antigen. 6 (a)-(f) show the surface of the immobilization layer 10, the primary antibody 11, the target bacteria (live bacteria) 12, the labeled substance 13, the labeled antigen 14, the light source 15, and the detector 16. Has been described.
[0068] 図 6において、まず、ノィォカラム用ガラス管に充填されたガラスビーズの固定ィ匕層 表面 10に一次抗体 11を非特異吸着させて固定ィ匕する（図 6 (a) )。なお、本工程の 詳細については、バイオカラム 2の作製工程において述べたとおりである。  In FIG. 6, first, the primary antibody 11 is non-specifically adsorbed on the surface 10 of the immobilization layer of the glass beads filled in the glass column for a Noyo column, and immobilization is performed (FIG. 6 (a)). The details of this step are as described in the preparation step of the biocolumn 2.
[0069] 次に、被検抗原を含む試料液の中に蛍光試薬を添加することで、標的菌となる生 菌 12を発光させる（図 6 (b) )。より具体的には、 CFDA希釈液が試料液の中に添カロ されると、被検抗原のうちの生菌は、細胞内 pHインジケーターたる CFDA (標識ィ匕物 質 13)を吸収し、加水分解によって蛍光を発するようになる。すなわち、 CFDAは、 生菌染色薬としての機能を有することになる。なお、試料液の中に CFDA希釈液を 添加した後、攪拌装置による攪拌によって、生菌による加水分解を促進することとし てもよい。これにより、より短時間かつ確実に、生菌に CFDAを吸収させることができ 、ひいては検査の迅速ィ匕に資することとなる。  Next, by adding a fluorescent reagent to the sample solution containing the test antigen, the bacterium 12 as a target bacterium is caused to emit light (FIG. 6 (b)). More specifically, when the diluted CFDA solution is added to the sample solution, live bacteria among the test antigens absorb CFDA (labeling substance 13), which is an intracellular pH indicator, and hydrolyze. It becomes fluorescent by decomposition. That is, CFDA has a function as a viable stain. After the CFDA diluent is added to the sample solution, the hydrolysis by viable bacteria may be promoted by stirring with a stirrer. As a result, CFDA can be absorbed into living bacteria in a shorter time and more reliably, thereby contributing to quick inspection.
[0070] 次に、被検抗原 (標識ィ匕抗原 14を含む）が存在する試料液を、バイオカラム 2の固 定化層表面 10に接触させ、一次抗体 11との抗原抗体反応によって被検抗原を捕捉 する（図 6 (c) )。そして、被検抗原の捕捉した後、リン酸緩衝液などの洗浄溶液をバイ ォカラム 2に流し込み、不純物や未反応一次抗体等を除去することで、被検抗原の 凝縮ィ匕 (濃縮化)及び被検抗原の固定ィ匕を行う（図 6 (d) )。なお、図 6 (b)に示す攪 拌工程と、図 6 (c)に示す捕捉工程及び図 6 (d)に示す固定ィ匕工程と、を複数回繰り 返し実行するのが好ましい。これにより、未反応状態の被検抗原の数が少なくなり、 ひいては検査精度 '検査感度の向上に寄与することとなる。 [0071] 次に、一次抗体 11によって固定ィ匕され、標識化抗原 14を含む被検抗原を、アル力 リ水溶液によって溶菌'抽出する（図 6 (e) )。このとき、循環経路内容量やフローセル 容量を少なくし、溶菌*抽出に必要なアルカリ水溶液の量を減少させることで、溶菌抽 出液中の菌濃度を高めることができ、ひいては検出感度を向上させることができる。 なお、本発明の実施の形態では、高濃度アルカリ水溶液を用いた力 例えば酸性緩 衝液ゃ界面活性剤などを併用することによって、より迅速かつ確実に被検抗原を溶 菌'抽出することができる。 Next, a sample solution containing the test antigen (including the labeled antigen 14) is brought into contact with the surface 10 of the immobilized layer of the biocolumn 2, and the test solution is subjected to an antigen-antibody reaction with the primary antibody 11. Capture the antigen (Fig. 6 (c)). After capturing the test antigen, a washing solution such as a phosphate buffer is poured into the biocolumn 2 to remove impurities, unreacted primary antibodies, etc., thereby condensing (concentrating) the test antigen and The test antigen is fixed (Fig. 6 (d)). Preferably, the stirring step shown in FIG. 6 (b), the capturing step shown in FIG. 6 (c), and the fixing step shown in FIG. 6 (d) are repeatedly performed a plurality of times. As a result, the number of unreacted test antigens is reduced, which contributes to the improvement of test accuracy and test sensitivity. Next, the test antigen immobilized by the primary antibody 11 and containing the labeled antigen 14 is lysed and extracted with an aqueous solution of Arikari (FIG. 6 (e)). At this time, the concentration in the lysate extract can be increased by reducing the volume in the circulation path and the flow cell, and by reducing the amount of alkaline aqueous solution required for lysis * extraction, thereby improving detection sensitivity. be able to. In the embodiment of the present invention, a test antigen can be lysed and extracted more quickly and reliably by using a force using a high-concentration alkaline aqueous solution, for example, an acidic buffer solution / surfactant in combination. .
[0072] 最後に、標識ィ匕抗原 14を、光源 15に対向して配置された検出器 16によって光学 的に検出する（図 6 (f) )。より具体的には、標識化物質 13を含む標識化抗原 14は、 光源 15より発せられた紫外線励起光によって蛍光を発し、集光レンズを備える検出 器 16においてこれを受光することで、電気信号 (クロマト信号)を取り出す。この電気 信号を測定'分析することによって、標識ィ匕抗原 14 (標的菌 12)を光学的に検出する ことができる。なお、本発明の実施の形態では、蛍光分光光度計を用いることとした 力 例えばパーティクルカウンタ一等の検出器を用いるなど、検出態様の如何は問わ ない。  Finally, the labeled antigen 14 is optically detected by the detector 16 arranged opposite to the light source 15 (FIG. 6 (f)). More specifically, the labeled antigen 14 containing the labeling substance 13 emits fluorescent light by ultraviolet excitation light emitted from the light source 15 and is received by the detector 16 having a condenser lens to generate an electric signal. (Chromatographic signal). By measuring and analyzing the electric signal, the labeled antigen 14 (target bacterium 12) can be optically detected. In the embodiment of the present invention, the detection mode does not matter, for example, a force using a fluorescence spectrophotometer. For example, a detector such as a particle counter is used.
[0073] 以上説明したように、図 6 (a)—図 6 (f)に示す一連の工程によれば、従来のサンド イッチ法よりも短時間で微生物検査を行うことができる。すなわち、従来のサンドイツ チ法では、被検抗原の検出までに 2回の抗原抗体反応が必要であつたが（図 12 (b) , (c)参照）、本発明によれば、標識化抗原 14と一次抗体 11との 1回の抗原抗体反 応で済むことから（図 6 (c)参照）、その分検査時間を短縮ィ匕することができ、ひいて は検査の迅速ィ匕を図ることができる。  As described above, according to the series of steps shown in FIGS. 6A to 6F, a microorganism test can be performed in a shorter time than in the conventional sandwich method. That is, in the conventional San Deutsch method, two antigen-antibody reactions were required before detection of the test antigen (see FIGS. 12 (b) and 12 (c)). Since only one antigen-antibody reaction between the antigen 14 and the primary antibody 11 is required (see FIG. 6 (c)), the test time can be reduced by that much, and the test can be performed quickly. Can be planned.
[0074] [変形例]  [Modification]
図 7は、本発明の他の実施の形態に係る検出装置の外観図である。主な特徴とし ては、特定の標的菌を捕捉可能なノィォカラム 65, 66を 2本設けている点である。な お、図 7では、 2本のバイオカラム 65, 66を設けることとした力 本発明はこれに限ら れず、例えば 3本以上のバイオカラムを設けることとしてもよい。複数本のバイオカラ ムを設けることによって、複数種類の標的菌を同時に検出することができるようになる [0075] 図 7において、本発明の他の実施の形態に係る検出装置は、各機器 'ポンプ'ボト ル等が 35± 1°Cの恒温槽（図中の四角枠)の内部に設置され、各機器やポンプは、 流路制御用シーケンサ 69で最適に制御される。恒温槽の内部には、標的菌を含む 試験試料供給槽 (試料ホツバ) 61、試料を攪拌する攪拌装置 (マグネスチックスター ラ） 62、不純物を取り除くろ過フィルター 63、流路を適宜切り替える流路切り替えバ ルブ 64、表面に標的菌抗体を固定ィ匕した微粒子ガラスが充填されたバイオカラム 65 , 66、試料を流動せしめる循環ポンプ 67、標的菌を光学的に検出する高感度蛍光 検出器 68が設置され、また、洗浄液の入ったボトル Bl l、固定ィ匕液の入ったボトル B 12、捕捉した染色菌の溶菌抽出液の入ったボトル B13が設置されている。以下、図 7に示す検出装置を用いた検査工程について概説する。 FIG. 7 is an external view of a detection device according to another embodiment of the present invention. The main feature is that two Noo columns 65 and 66 that can capture specific target bacteria are provided. In FIG. 7, the force of providing two biocolumns 65 and 66 is not limited to this. For example, three or more biocolumns may be provided. Providing multiple biocolumns enables simultaneous detection of multiple types of target bacteria In FIG. 7, in the detection device according to another embodiment of the present invention, each device such as a 'pump' bottle is installed inside a constant temperature bath (square frame in the drawing) at 35 ± 1 ° C. The respective devices and pumps are optimally controlled by the flow path control sequencer 69. Inside the constant temperature bath, a test sample supply tank containing the target bacteria (sample hopper) 61, a stirrer for stirring the sample (magnetic stirrer) 62, a filtration filter 63 for removing impurities, and a flow path switch for switching the flow paths appropriately A valve 64, biocolumns 65 and 66 filled with fine particle glass with the target bacteria antibody fixed on the surface, a circulating pump 67 for flowing the sample, and a high-sensitivity fluorescence detector 68 for optically detecting the target bacteria are installed. In addition, a bottle B11 containing a washing solution, a bottle B12 containing a fixative solution, and a bottle B13 containing a lysate of a captured stained bacterium are provided. Hereinafter, the inspection process using the detection device shown in FIG. 7 will be outlined.
[0076] まず、規定量の試料を定法によってストマツキングし、試験溶液（50— 100ml)を試 験試料供給槽 61に入れる。そして、攪拌装置 62で攪拌しながら蛍光染色試薬として の CFDAを添加し、生菌を染色する。約 10分間の攪拌の後、ろ過フィルター 63を通 して不純物を取り除くとともに、試料流路 (バイオカラム 65)へ導入する。なお、流路 切り替えバルブ 64をフィルター逆洗浄系に切り替え、ろ過フィルター 63を洗浄してお First, a specified amount of a sample is striked by a standard method, and a test solution (50 to 100 ml) is put into a test sample supply tank 61. Then, while stirring with the stirrer 62, CFDA as a fluorescent staining reagent is added to stain live bacteria. After stirring for about 10 minutes, the impurities are removed through the filtration filter 63 and introduced into the sample channel (biocolumn 65). Switch the flow path switching valve 64 to the filter back washing system, and wash the filtration filter 63.
<o <o
[0077] 次いで、ろ過ユニット（ろ過フィルター 63)を通過した試験溶液は、バイオカラム 65, 66を通過し、全流路洗浄系流路を循環することで、数回、バイオカラム 65, 66内を 通過する。その後、固定化抗体に捕捉された染色菌体 (標識化抗原）は、ボトル B13 力もバイオカラム 65, 66内のリサイクル系流路に少量に添加された溶菌抽出液を数 回リサイクルすることによって、高濃度に抽出される。抽出が完了した試験溶液は、バ ィォカラム 65, 66の下部の切り替えバルブ 64によって、高感度蛍光検出器 68へと 導入され、電気信号としてクロマトグラムが描画される。  [0077] Next, the test solution that has passed through the filtration unit (filtration filter 63) passes through the biocolumns 65 and 66, and circulates through all the washing channels in the biocolumns 65 and 66. Pass through. After that, the stained bacterial cells (labeled antigen) captured by the immobilized antibody were re-used several times by recycling the lysed extract added in small amounts to the recycling channel in the biocolumns 65 and 66 using the bottle B13. Extracted to high concentration. The test solution from which extraction has been completed is introduced into the high-sensitivity fluorescence detector 68 by the switching valve 64 below the biocolumns 65 and 66, and a chromatogram is drawn as an electric signal.
[0078] より具体的には、抽出が完了した試験溶液は、高感度蛍光検出器 68のフローセル に導入され、その試料溶液のうちの染色菌体は、光源から発せられた紫外線励起光 によって蛍光を発する。そして、その蛍光を集光レンズで受光し、光信号を電気信号 に変換することによって、クロマトグラムが描画される。  [0078] More specifically, the test solution from which extraction has been completed is introduced into the flow cell of the high-sensitivity fluorescence detector 68, and the stained bacterial cells in the sample solution are fluoresced by ultraviolet excitation light emitted from the light source. Emits. Then, the fluorescent light is received by a condenser lens, and an optical signal is converted into an electric signal, whereby a chromatogram is drawn.
[0079] 最後に、溶菌'抽出が終了した後に、標的菌抗体を固定ィ匕した微粒子ガラスが充 填されたバイオカラム 65, 66は、ボトル B11内の洗浄液によって洗浄され、リフレツシ ュされる。 [0079] Finally, after the completion of the lyse extraction, the fine particle glass on which the target bacterium antibody is immobilized is filled. The packed biocolumns 65 and 66 are washed with the washing solution in bottle B11 and refreshed.
[0080] 以上概説したように、図 7に示す検出装置によれば、 2本のバイオカラム 65, 66を 設けることで、 2種類の標的菌を 1回の検査の流れの中で同時に捕捉することが可能 となり、ひいては検査の効率化'迅速ィ匕に寄与することとなる。  As outlined above, according to the detection device shown in FIG. 7, by providing two biocolumns 65 and 66, two types of target bacteria are simultaneously captured in one test flow. It is possible to contribute to the improvement of the efficiency of the inspection and the speed of the inspection.
[0081] なお、抗体の異なる複数のノィォカラムを直列に設置することで、複数の標的菌を 同時に検出することができる。また、同じ抗体のバイオカラム複数を並列に設置する ことで、特定の標的菌における複数検水を同時に検出することができる。さらに、この 両者を併用することも可能である。  [0081] By installing a plurality of Noo columns with different antibodies in series, a plurality of target bacteria can be detected simultaneously. In addition, by installing multiple biocolumns of the same antibody in parallel, it is possible to simultaneously detect multiple samples of a specific target bacterium. Further, both can be used in combination.
[0082] [培養工程]  [Culture step]
本発明の実施の形態に係る検出方法では、基本的に培養工程がなくても十分に標 的菌を検出することができる。しかし、必要に応じて、標的菌を培養することで、より確 実な検査結果を得ることができる。培養工程は、例えば、検出装置にヒータを備えて おき、固定ィ匕工程前に培養を施すか、或いは、検査工程が組み込まれている検出装 置全体を一定温度にして培養を施すことで実行することができる。  The detection method according to the embodiment of the present invention can sufficiently detect a target bacterium without a culturing step. However, more reliable test results can be obtained by culturing the target bacteria as necessary. The culturing step is performed, for example, by providing a heater in the detection device and performing culturing before the fixing step, or by culturing the entire detection device in which the inspection process is incorporated at a constant temperature. can do.
実施例 1  Example 1
[0083] 図 8は、図 3に示す流路系において、バイオカラム 2の性能実験を行った際の測定 結果を示す図である。より具体的には、大腸菌（E.coli)の注入量 (CFUZlOOml)に 対する CFDA蛍光強度を示している。図 8に示す表によれば、大腸菌注入量と、溶 菌抽出液中の CFDA蛍光強度との間には、高い相関が見られることが分かる。すな わち、図 8に示す表の各データを 2次元フィールドにプロットした図 9によれば、大腸 菌の注入量が増加するにつれて、 CFDA蛍光強度も増加しており、標的菌が適切に 検出できたことが分力る。  FIG. 8 is a diagram showing the measurement results when a performance experiment of the biocolumn 2 was performed in the flow channel system shown in FIG. More specifically, it shows the CFDA fluorescence intensity with respect to the injection amount (CFUZlOOml) of E. coli. According to the table shown in FIG. 8, it can be seen that a high correlation is observed between the amount of E. coli injected and the fluorescence intensity of CFDA in the lysate extract. In other words, according to FIG. 9, in which each data in the table shown in FIG. 8 is plotted in a two-dimensional field, as the injection amount of E. coli increases, the CFDA fluorescence intensity increases, and the It helps to detect that.
[0084] ここで、図 8に示す表において、大腸菌の流入量が最小濃度の lOCFUZmlの検 水に対しても、比較的強い蛍光スペクトル (平均 1. 2050)が得られていることから、 約 5CFUZmlの検水に対しても適切な標的菌検出が可能であると考えられる。また 、循環経路内の容量やミクロフローセル容量を少なくし、溶菌抽出液の必要量を減少 させることによって、溶菌抽出液の菌濃度を高め、約約 1一 5CFUZmlの検水に対 しても適切な標的菌検出が可能であると考えられる。 [0084] Here, in the table shown in Fig. 8, a relatively strong fluorescence spectrum (average 1.2050) was obtained even for a sample of lOCFUZml with a minimum concentration of Escherichia coli. It is considered that the target bacteria can be detected appropriately even for the 5CFUZml sample. In addition, by reducing the volume in the circulation path and the volume of the micro flow cell, and reducing the required amount of lysate extract, the bacterial concentration of the lysate extract is increased, and it is necessary to test about 15 to 15 CFUZml of water. Therefore, it is considered that appropriate target bacteria can be detected.
[0085] 図 10は、図 3に示す流路系において、バイオカラム 2の性能実験を行った際の測定 結果を示す表である。特に、図 10 (a)は、染色処理を施した死菌に対する CFDA蛍 光強度を示している。図 10 (a)に示す表によれば、被検抗原のうちの死菌を注入し ても、溶菌抽出液中の CFDA蛍光強度は非常に微弱であることが分かる。すなわち 、実際の検水中に、直接食中毒被害をもたらさない死菌が混在していたとしても、干 渉されずに高 、精度で生菌を評価することができ、ひ 、ては死菌のみを含む適合品 を排除してしまうといった問題を解決できることが分かる。  FIG. 10 is a table showing measurement results when a performance experiment of the biocolumn 2 was performed in the flow channel system shown in FIG. In particular, FIG. 10 (a) shows the CFDA fluorescence intensity for the dead bacteria subjected to the staining treatment. According to the table shown in Fig. 10 (a), even when dead bacteria among the test antigens were injected, the CFDA fluorescence intensity in the lysed extract was very weak. That is, even if dead bacteria that do not directly cause food poisoning are mixed in the actual test water, viable bacteria can be evaluated with high accuracy without any interference, and only dead bacteria can be detected. It can be seen that the problem of eliminating conforming products, including, for example, can be solved.
[0086] また、図 10 (b)は、大腸菌（E.coli)以外の複数種類の菌（大腸菌群： C.freundii、腸 内細菌科菌： S.marcescens)の注入量（CFUZlOOml)に対する CFDA蛍光強度を 示している。図 10に示す表によれば、大腸菌以外の菌に対しては、溶菌抽出液の C FDA蛍光強度は弱いことが分かる。すなわち、死菌と同様に、試料溶液の中に標的 菌以外の菌が共存している場合であっても、その影響は比較的低くすることができる  [0086] Fig. 10 (b) shows CFDA versus injection amount (CFUZlOOml) of multiple types of bacteria other than E. coli (E. coli: C. freundii, Enterobacteriaceae: S. marcescens). Shows the fluorescence intensity. According to the table shown in FIG. 10, it can be seen that the lysis extract has a low CFDA fluorescence intensity for bacteria other than E. coli. In other words, similarly to dead bacteria, even when bacteria other than the target bacteria coexist in the sample solution, the effect can be relatively low.
[0087] 図 11は、図 3に示す流路系において、繰り返し使用によるバイオカラム 2の性能実 験を行った際の測定結果を示す表である。より具体的には、バイオカラム 2の累積使 用回数（回）に対する CFDA蛍光強度を示している。図 11によれば、バイオカラム 2 を累積的に 2回使用すると、 2回目の使用時には、溶菌抽出液中の CFDA蛍光強度 が約 98%減少することが分かる。これは、標的菌の抽出に、溶菌作用のある高濃度 のアルカリを使用しており、この際に、菌とともにタンパク質である抗体にもダメージを 与えていると考えられる。従って、例えば抗体へのダメージが少ない溶菌抽出液を用 いることで、バイオカラム 2の複数回使用が可能になる。 FIG. 11 is a table showing measurement results when performance tests of the biocolumn 2 by repeated use were performed in the flow channel system shown in FIG. More specifically, it shows the CFDA fluorescence intensity with respect to the cumulative number of uses (times) of the biocolumn 2. According to FIG. 11, it can be seen that when the biocolumn 2 is used twice cumulatively, the CFDA fluorescence intensity in the lysed extract decreases by about 98% at the second use. This is because a high concentration of alkali having a lytic action is used for extraction of the target bacterium. At this time, it is considered that the antibody, which is a protein, is damaged together with the bacterium. Therefore, for example, by using a lysate extract that causes less damage to the antibody, the biocolumn 2 can be used multiple times.
[0088] 次に、試験材料の種類や量を変えて行った評価試験にっ 、て、以下に概説する。 Next, an outline of an evaluation test performed by changing the type and amount of the test material will be described below.
[0089] まず、 MPN法等によって菌数が推定されている菌液 100mlを試験装置の試料供 給瓶に移し、 CFDAを含む生菌染色液を lml加え、その全量を毎分 10mlの流速で バイオカラムに 2回循環させた後、排水する。そして、試料液の全量をバイオカラムに 適用した後、適量のバイオカラム洗浄液を流し、バイオカラムの内部を洗浄した後、 流路を切り替えて、バイオカラム内部に残ったバイオカラム洗浄液をすベて排出する [0090] 次いで、上述の工程で生菌染色され、バイオカラムにトラップされた標的菌を全量 1 Omlの溶菌抽出液を用いて溶菌抽出しながら、蛍光分光光度計のフローセルに導入 し、蛍光強度の測定を行う。そして、その測定の終了後、滅菌リン酸緩衝希釈水を用 V、て全ての流路系を洗浄する。 First, 100 ml of the bacterial solution whose number of bacteria is estimated by the MPN method or the like is transferred to a sample supply bottle of a test device, and 1 ml of a viable bacterial staining solution containing CFDA is added. After circulating twice through the biocolumn, drain. Then, after applying the entire amount of the sample solution to the biocolumn, an appropriate amount of the biocolumn washing solution is flowed, and after the inside of the biocolumn is washed, the flow path is switched and all the biocolumn washing solution remaining inside the biocolumn is removed. Discharge [0090] Then, the target bacteria stained with live bacteria in the above-described steps and trapped in the biocolumn were lysed and extracted using a lysis extract of a total amount of 1 Oml, and introduced into the flow cell of the fluorescence spectrophotometer. Measurement. Then, after the measurement is completed, all the channel systems are washed with sterile phosphate buffered diluted water V.
[0091] 以上が評価試験の概要であるが、要した時間は約 1時間である。力かる評価試験 において、試料中に標的生菌が約 30CFU以上存在すれば、十分に検出する能力 力 Sあることが分力つた。また、大腸菌以外の菌 (大腸菌群や腸内細菌)の影響、死菌 の影響は極めて軽微であり、実用上問題はないことも分力つた。従って、本発明では 、対象を大腸菌として例示しているが、抗原抗体反応によって捕集できるものであれ ばいかなるものであってもよぐ例えば、力ビ類も対象とすることができる。  [0091] The above is the outline of the evaluation test, and the time required is about one hour. In a powerful evaluation test, it was concluded that if the target viable cells were present in the sample in an amount of about 30 CFU or more, they had sufficient detection ability S. In addition, the effect of bacteria other than Escherichia coli (such as coliform bacteria and intestinal bacteria) and the effects of killed bacteria were extremely slight, and it was also a factor that there was no practical problem. Therefore, in the present invention, the subject is exemplified as Escherichia coli, but any object can be used as long as it can be collected by an antigen-antibody reaction.
産業上の利用可能性  Industrial applicability
[0092] 本発明に係る検出方法及び検出装置は、被検抗原に、その被検抗原内の生菌に よって酵素分解される標識化物質を作用させた標識化抗原を検出することで、試料 溶液の生菌を標的菌とすることができ、また、検査の迅速性及び確実性を担保し得る ものとして有用である。 [0092] The detection method and the detection device according to the present invention detect a labeled antigen obtained by allowing a labeled substance that is enzymatically decomposed by viable bacteria in the test antigen to act on the test antigen, thereby detecting the sample. The viable bacteria in the solution can be used as target bacteria, and are useful as those that can ensure quickness and certainty of the test.
図面の簡単な説明  Brief Description of Drawings
[0093] [図 1]本発明の実施の形態に係る検出装置の外観図である。 FIG. 1 is an external view of a detection device according to an embodiment of the present invention.
[図 2]本発明の実施の形態に係る検出装置に設置されたバイオカラムの拡大図であ る。  FIG. 2 is an enlarged view of a biocolumn installed in the detection device according to the embodiment of the present invention.
[図 3]図 1に示す検出装置を用いて微生物検査を行う際の流路系統図である。  FIG. 3 is a flow path diagram when performing a microorganism test using the detection device shown in FIG. 1.
[図 4]図 3に示す流路系統図における検査工程の概略を示すフローチャートである。  FIG. 4 is a flowchart showing an outline of an inspection process in the flow path diagram shown in FIG. 3.
[図 5]固定相を効果的に攪拌する様子を示す説明図である。  FIG. 5 is an explanatory view showing how a stationary phase is effectively stirred.
[図 6]本発明の実施の形態に係る検出方法の主要工程を示す模式図である。  FIG. 6 is a schematic view showing main steps of a detection method according to an embodiment of the present invention.
[図 7]本発明の他の実施の形態に係る検出装置の外観図である。  FIG. 7 is an external view of a detection device according to another embodiment of the present invention.
[図 8]図 3に示す流路系において、バイオカラムの性能実験を行った際の測定結果を 示す図である。  FIG. 8 is a view showing measurement results when a performance experiment of a biocolumn was performed in the flow channel system shown in FIG. 3.
[図 9]図 8に示す表の各データを 2次元フィールドにプロットした図である。 [図 10]図 3に示す流路系において、バイオカラムの性能実験を行った際の測定結果 を示す表である。 FIG. 9 is a diagram in which each data of the table shown in FIG. 8 is plotted in a two-dimensional field. FIG. 10 is a table showing measurement results when performing a performance experiment on a biocolumn in the flow channel system shown in FIG. 3.
[図 11]図 3に示す流路系にお 、て、繰り返し使用によるバイオカラムの性能実験を行 つた際の測定結果を示す表である。  FIG. 11 is a table showing measurement results of performance tests of a biocolumn by repeated use in the flow channel system shown in FIG. 3.
[図 12]従来のサンドイッチ法の主要工程を示す図である。  FIG. 12 is a view showing main steps of a conventional sandwich method.
符号の説明 Explanation of symbols
1 検出装置  1 Detector
2 バイ才力ラム  2 By Talent Ram
3 M— Cell  3 M—Cell
10 固定化層表面  10 Immobilization layer surface
11 一次抗体  11 Primary antibody
12 標的菌 (生菌）  12 Target bacteria (live bacteria)
13 標識化物質  13 Labeling substance
14 標識化抗原  14 Labeled antigen
15 光源  15 light source
16 検出器  16 detector

Claims

請求の範囲 The scope of the claims
[1] 被検抗原と、前記被検抗原内の生菌によって酵素分解される標識化物質と、の作 用によって、前記被検抗原内の生菌を特異的に標識ィ匕して検出する検出方法であ つて、  [1] The action of a test antigen and a labeled substance that is enzymatically degraded by live bacteria in the test antigen specifically detects and detects live bacteria in the test antigen. Detection method,
前記被検抗原に前記標識化物質を作用させて光学的に検出可能な標識化抗原を 生成し、  Reacting the labeled substance with the test antigen to produce an optically detectable labeled antigen;
前記被検抗原に特異的に結合しうる特異的結合抗体を固定ィ匕してなる固定相にお The stationary phase is formed by immobilizing a specific binding antibody capable of specifically binding to the test antigen.
V、て、前記標識化抗原を捕捉することを特徴とする検出方法。 V. A detection method comprising capturing the labeled antigen.
[2] 前記固定相において、増殖培養液によって増殖した前記標識ィ匕抗原を捕捉するこ とを特徴とする請求項 1記載の検出方法。 [2] The detection method according to [1], wherein the labeled phase captures the labeled antigen grown by a growth medium.
[3] 前記標識化抗原を含む試料溶液を複数回循環させながら、前記固定相において、 循環される標識化抗原を捕捉することを特徴とする請求項 1又は 2記載の検出方法。 3. The detection method according to claim 1, wherein the circulating labeled antigen is captured in the stationary phase while circulating the sample solution containing the labeled antigen a plurality of times.
[4] 複数種類の前記被検抗原のそれぞれに対応して特異的に結合しうる特異的結合 抗体を固定ィ匕してなる複数種類の固定相において、前記被検抗原を捕捉することを 特徴とする請求項 1から 3記載の検出方法。 [4] The test antigen is captured in a plurality of types of stationary phases formed by immobilizing a specific binding antibody capable of specifically binding to each of the plurality of types of test antigens. 4. The detection method according to claim 1, wherein:
[5] 被検抗原に特異的に結合しうる特異的結合抗体を固体ィ匕してなる固定相を設置可 能なカラムを有する検出装置であって、 [5] A detection device having a column capable of installing a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen,
前記固定相が設置されたカラムにおいて、前記被検抗原が標識化された標識化抗 原を捕捉することを特徴とする検出装置。  A detection device, wherein the test antigen captures a labeled antigen labeled with the test antigen in a column provided with the stationary phase.
[6] 前記検出装置は、さらに、液体を攪拌する攪拌装置を備え、 [6] The detection device further includes a stirring device for stirring the liquid,
前記標識化抗原は、前記攪拌装置にお!ヽて標識化されることを特徴とする請求項 The labeled antigen is labeled by the stirring device.
5記載の検出装置。 The detection device according to 5.
[7] 前記カラムは、複数回使用可能であることを特徴とする請求項 5記載の検出装置。  7. The detection device according to claim 5, wherein the column can be used a plurality of times.
[8] 被検抗原に特異的に結合しうる特異的結合抗体を固体ィ匕してなる固定相を設置可 能なカラムを複数有する検出装置であって、 [8] A detection device having a plurality of columns capable of setting a stationary phase formed by solid-binding a specific binding antibody capable of specifically binding to a test antigen,
前記固定相が設置された複数のカラムにおいて、前記被検抗原が標識化された標 識化抗原を捕捉することを特徴とする検出装置。  A detection device, wherein the plurality of columns provided with the stationary phase capture a labeled antigen in which the test antigen is labeled.
[9] 被検抗原に特異的に結合しうる特異的結合抗体を固定化してなる固定相が設置さ れたノ ィ才力ラム。 [9] A stationary phase consisting of immobilized specific binding antibodies capable of specifically binding to the test antigen is provided. No talent ram.
被検抗原に特異的に結合しうる特異的結合抗体を固定ィ匕してなる固定相が設置さ れたバイオカラムにぉ 、て、  A biocolumn provided with a stationary phase formed by immobilizing a specific binding antibody capable of specifically binding to a test antigen,
前記ノィォカラム内の圧力変動を利用して、前記固定相を攪拌する方法。  A method in which the stationary phase is agitated by utilizing pressure fluctuation in the Noo column.
PCT/JP2005/003584 2004-04-28 2005-03-03 Method and apparatus for detection of live bacterium within test subject antigen through specifically labeling thereof WO2005106454A1 (en)

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